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Saturday, October 3, 2015

Functional MRI of dolphins?

Those of you who follow me on Twitter may have noticed that I've been scanning some post mortem brains of Cetacea over the past year or so. That's whales, dolphins and porpoises to you and me. The brains come in all shapes and sizes, from a rather tiny Amazon river dolphin, about the size of a fist, to fin and sei whale brains that are so wide they have to be inserted sideways, hemisphere-first, into the 3 T (human) head coil. The conditions and ages of the brains vary tremendously as well. Some have been fixed in formaldehyde for decades yet yield remarkably decent signal, others have been stored in ethanol and are as hard as rubber with T2 to match. A few months ago we obtained a recently deceased fresh brain of a white-sided dolphin which we were able to scan within about twelve hours of its demise. The image quality was magnificent.

What do we plan to do with all the post mortem data? That is still being formulated. Initial motivation for the project came from some Berkeley anthropologists with an interest in comparative neuroanatomy across higher mammalian species. Coincidentally, Greg Berns' group at Emory has recently produced a nice example of dolphin brain tractography and his recent study is a good example of what might be done in future. There's a commentary on Greg's study here, and an example image from his paper below. We are now determining how we might combine resources, share data and all that good stuff. More on what will be available to whom and when as we progress.

From: Berns et al.

In any case, after I posted the white-sided dolphin MRI to Twitter someone asked, likely facetiously (I suppose that should be flippantly), whether functional MRI was next. FMRI of dolphins was the subject of an April Fool's Day joke a few years ago, and it does seem far-fetched at first blush. So, too, does studying trained dogs with fMRI, but Greg Berns' team is already doing that. Since thought experiments are cheap I figured I'd write a blog post to consider what might be feasible today if one were sufficiently motivated (read sufficiently well funded) to want to do fMRI of cetaceans. If nothing else we might learn something as we're forced to consider the manifold factors.

What's been done before?

Like any good experiment, thought or otherwise, we should begin with a review of the literature. (A month in the lab will frequently save an hour in the library, as the old aphorism has it.) And it turns out that some clever folks at UCSD have done both PET and SPECT scanning of a dolphin in the last few years. It's not fMRI but it is functional brain imaging and they used structural MRI (at 0.5 T) to locate the activity. Here's a dolphin getting a structural MRI scan in this figure from their 2006 paper:

(From Ridgway et al., 2006. DOI: 10.1242/​jeb.02348)

Structural MRI has a more extensive literature than the functional stuff. If you search PubMed for "dolphin brain MRI" you get 19 publications, most of which concern post mortem specimens. Some brains were fresh and in situ, others were fixed. But the papers from UCSD that reported PET and SPECT results seem to be the only published academic studies using MRI of a live dolphin, too. There are reports of dolphins receiving MRI for diagnostic medical purposes, such as this one, but they show CT scanners in their illustrations.

Sizing the hardware and getting a dolphin-friendly "patient bed"

I shall assume we want to be able to scan an adult bottlenose dolphin. The main constraints are the mass of the animal and the size and location of the dorsal fin and pectoral flippers. Open style (stand-up) magnets accommodate the dorsal fin easily but the magnet strength is low. Likewise, open MRIs of the type used by UCSD (shown above) accommodate the pectoral fins but again the magnetic field is low. The best I've found is the new 1.2 T Oasis from Hitachi. An impressive field strength for an open MRI, but let's aim for at least 3 T assuming we want lots of fMRI contrast and high resolution anatomical scans.

A wide bore 3 T MRI might work for us but before we commit to what's on the market we need to consider the dimensions and mass of an adult dolphin in more detail. The following dimensions were estimated from a paper on dolphin growth rate together with a basic view of dolphin anatomy courtesy of the Texas Marine Mammal Stranding Network. Our model adult bottlenose dolphin is 2.7 m long. The distance from the center of its brain to the front of the dorsal fin is about a meter. This distance is compatible with Verio & Skyra magnets from Siemens. The Skyra has 70 cm usable bore diameter with 173 cm total length, meaning that the center of the magnet is 86.5 cm from the front flange of the magnet. The leading edge of the dorsal fin would end up just outside the magnet.

But what about that 70 cm bore? For a length of 2.7 m the girth anterior to the dorsal fin is about 1.9 m. This gives a diameter at the widest point of 61 cm assuming our dolphin is (a) slim n' trim and (b) well approximated by a cylinder. We probably shouldn't assume either of these to be true. Furthermore, the entire bore isn't usable because we need some sort of table or other support to move the animal in and out of the magnet, and make sure it's not uncomfortable during the scan. While a flat table as for humans might work given a sufficiently large magnet bore, for a dolphin it's not the ideal shape. If our dolphin is 61 cm across at the widest point and the magnet bore is 70 cm diameter then we only have 9 cm to play with. An adult dolphin is considerably heavier than your average human, too, at 200-250 kg. We're going to have to replace the patient table. Would 9 cm be enough space to accommodate some sort of curved bed/sled that could move the animal in and out? Perhaps, with suitably strong materials or with the magnet bore lowered relative to the surrounding floor, as happens for some very large, high field magnets.

Cross section through a modern superconducting MRI magnet, showing all the principal components except the plastic covers that provide the external facade as well as some acoustic noise attenuation.
From: Lvovsky, Stautner & Zhang, Supercond. Sci. Technol. 26 093001 (2013). doi:10.1088/0953-2048/26/9/093001

Beyond supporting the mass of the animal there is an additional qualifier to the curved "dolphin bed." As I'll discuss more below, dolphins are highly sensitive to vibration and hear the majority of sounds through bone conduction via their lower jaw. Thus, serious consideration would be needed for anti-vibration measures when selecting materials for our bed/sled. It's difficult to fully decouple a bed from the magnet, even with cantilevered designs, so some sort of anti-vibration liner is probably implied.

What about gradients, RF transmission and reception? Here we can extrapolate reasonably well from what we use today for humans. As shown in the above illustration, a magnet-sized (so-called body) RF transmit coil and gradients are already accounted for in a commercial 70 cm clear bore system such as a Skyra. If it transpires that a larger clear bore is needed, e.g. to accommodate the custom bed/sled, then one obviously needs larger, custom gradient and body RF transmit coils proportional to the larger magnet bore. These wouldn't be cheap but the engineering is entirely tractable.

Signal reception could be achieved by something as simple as a blanket coil. The dolphin's brain is located just behind its blowhole, as shown below. We obviously don't want the animal to suffocate in the MRI so we would need to modify the blanket coil for breathing. Alternatively, one might easily pursue a custom coil design that resembles a large bowl and incorporate sufficient space for the blowhole. There are many, many options when it comes to RF signal reception, depending on whether you want to do accelerated image acquisition, how the coil is to be held in place, etc.

Basic anatomy of the Atlantic Bottlenose Dolphin. From the Texas Marine Mammal Stranding Network website.

Health and welfare issues

In addition to the vibration, what about sound pressure levels through the air? These are a big deal for humans, and dolphins are known to hear frequencies almost an order of magnitude higher than humans. Hearing is arguably a dolphin's most sensitive sense. If one were to design and build a custom 3 T magnet with a larger bore - say a usable bore of 1 meter diameter - then there would be sufficient space to install aggressive acoustic and vibration damping. In the absence of a more refined solution, one simple option would be to construct a fiberglass bore liner onto which several different densities of acoustic foam can be attached. (We did this for an old 4 T magnet years ago and the reduction in sound pressure level was better than 20 dB in the bore. Adding extra foam to the backs of the magnet covers - the facade you actually see from the outside - also reduces echoes and reduces acoustic noise both in the bore and outside.) Anyway, the bottom line on acoustic noise and vibration is that much designing and testing would be needed to ensure that the final sound/vibration level was safe.

At the same time it is important to ensure the shape and surface of the "dolphin bed" is comfortable and doesn't place undue pressure on any part of the animal's body. With humans we've used memory foam on the patient bed of my Siemens 3 T since we realized that people fidget less when they're comfortable. Memory foams allow better distribution of weight to minimize pressure points. For an animal used to having its weight supported by water this issue becomes even more critical, especially if one is expecting to conduct a scan over twenty minutes or so.

Keeping MRI subjects cool is always important, both for safety and comfort, but it is paramount for an animal that experiences external water cooling naturally. Blowing air down the bore is okay for (some) humans in an MRI but it will tend to dry out the dolphin's skin. The bore could be lined instead with a water misting system to keep the animal moist as well as cool. The magnet would need to be drained appropriately, and there would need to be suitable precautions to ensure water doesn't get where it's not wanted, such as into connectors for an RF coil. Would the mist or water pooling on the bed/sled cause issues for the MR images themselves, e.g. due to signal aliasing? Probably not if the right RF coil is used for signal reception. A full bore "body" transmission coil is already heavily (electrically) loaded with a 200+ kg dolphin inside it; a few liters of water isn't going to matter much. On reception, if we're using a curved surface coil like a bowl or even a blanket coil then we really only need to worry about water pooling on the coil's upper surface, a situation that could be ameliorated with drainage if the shape doesn't fix the problem for us. And of course we can always turn the misters off when we're actually scanning.

How to approach fMRI of dolphins?

Physiology and BOLD

The goal here is functional MRI. Do we expect dolphin brains to exhibit BOLD contrast as we are used to seeing it in humans? On the one hand there is an abundance of evidence that mammalian brains exhibit rather consistent BOLD responses to stimuli. Studies of monkeys, dogs, cats and rodents have all shown robust BOLD contrast, whether awake or anesthetized. (Dead fish? Not so much.) We also have the UCSD studies using PET and SPECT as evidence that cerebral blood flow (CBF) might be estimated using the same methods in dolphins as for humans. So the chances of getting measurable BOLD and/or CBF changes in response to tasks would seem to be pretty good.

Cetaceans have evolved to dive and may be able to endure hypoxia for long periods compared to land-based mammals like us. Might this ability affect the BOLD contrast in interesting ways? Bottlenose dolphins generally have higher hematocrit and red blood cell levels than us land-lubber humans, unless we are high-altitude endurance athletes. And, just like us, the animal's fitness is a prime determinant of its blood constituents so we would want to record as many physiologic parameters as we can in order to distinguish neurovascular changes from concomitant effects. Variable hematocrit has already been implicated as an important experimental factor when studying exercising marine mammals. We're not going to have a dolphin diving in our MRI of course, but the dolphin's exercise history and general fitness may become crucial if we expect to compare fMRI results across multiple sessions. Definitely lots of factors to bear in mind when designing the fMRI experiment.

As for effects of the magnetic field, there is recent evidence that bottlenose dolphins can detect magnetism in otherwise identical lumps of metal concealed in opaque plastic barrels suspended in the water. But other than approaching barrels containing magnetic material more quickly, there was no difference in subsequent behavior: "The fact that all other behaviours did not differ between magnetized and control stimulus may reflect that magnetic fields are neither particularly attractive nor repulsive to dolphins." That's good. What we don't know, of course, is whether the strong magnetic field of an MRI would induce the dolphin equivalent of vertigo or other sensations that humans experience routinely.

Training for and conducting an fMRI experiment

I'm sure that most of you will have considered the ethics of attempting fMRI of a dolphin well before this point. I have neglected it until now simply because I wanted to consider the practical hurdles first. If we had run into an insurmountable problem then the ethics, while interesting, become moot. At the point of wanting to conduct an actual experiment, however, the ethics of the enterprise loom large and unavoidable.

Let's set a few limits. I'll assume that we want to scan animals that are available only because of extenuating circumstances. We are not going to attempt to scan wild dolphins unless by some miracle a wild dolphin can be coaxed into a scanner and lay still for the duration without significant assistance. Seems unlikely, even if an MRI scanner were partly submersed off a dock in Santa Cruz and a dolphin could swim right up into it. So we are talking about attempting to use dolphins in captivity only. I don't particularly relish the thought of dolphins in captivity for any reason, but I do understand that there are times when dolphins end up there and it may be inappropriate to release them. For example, some animals may have been rescued as orphaned youngsters or born into captivity and so lack the survival skills of an adult wild dolphin. Others may have been injured to the point where survival in the wild is unlikely. No doubt this would be the most contentious aspect of doing fMRI of a dolphin so I'll leave the ethics for you to debate in the comments below.

For the purposes of this thought experiment I shall assume that we have an adult dolphin living in captivity through no fault of its own. Furthermore, I shall assume that a committee of suitably qualified folks has determined that the perceived benefits of scanning the animal are sufficient to overcome objections and risks, just as we decide when studying any vulnerable population. As we move on to consider the training required for a dolphin fMRI experiment we will need to think carefully about the types of stimuli and responses we might want to use and determine the limitations imposed.

Perhaps the simplest fMRI experiment would be the "resting state." Here we might conceive of training a dolphin to lie still in a tube for ten minutes in exchange for a reward, such as Craig Bennett's dead salmon. This might permit discovery of the intrinsic connectivity in the dolphin's brain. Task-based fMRI would involve a far higher degree of training, but with a suitable mock scanner and enough fish rewards there would be a good chance of success if what's already been achieved with dogs is any guide.

In most human fMRI studies, and in the earliest dog fMRI studies, the stimuli are visual. Is that a good choice for a dolphin? Since I'm not a marine mammal expert I have no idea. What I do know, however, is that a dolphin's eyes are more on the sides of its head than forward-looking, as for humans and dogs. If we are going to use visual stimuli then we need to be thinking about the binocular visual abilities of the dolphin, perhaps resorting to twin goggles for monocular or binocular display, or a single target mounted sufficiently far from the dolphin's nose for it to see with both eyes.

Other stimuli might be possible. Those clever people at UCSD came up with a device that permits a dolphin to echolocate while out of water. The echolocation clicks "produced by the dolphin are detected with a hydrophone embedded in a suction cup on the melon." For stimuli: "The experimental approach relies on the generation of electronic, or “phantom” echoes, rather than the use of a physical target directly ensonified by the animal." How does the dolphin sense the echoes? "The echo signals are then projected to the dolphin via a transducer placed on the lower jaw." Here's a schematic of the echolocation pathway for a dolphin, taken from the Wikipedia page on animal echolocation:

Sound generation, propagation and reception in a toothed whale. From the Wikipedia page on animal echolocation.

That would surely be the hard part done, especially as the melon is forward of the brain and blowhole. When mounted the device ought not be in conflict with a receive RF coil. Making the device MR compatible ought to be straightforward by comparison to inventing it in the first place! Would it be possible to use this device during scanning while ensuring good attenuation of the scanner's vibrations and noises? The scanner peak acoustic frequencies are typically less than 3 kHz whereas dolphins echolocate from 40-150 kHz. Even allowing for a few harmonics there should be minimal energy produced by the scanner in that range.

The whistles and clicks that humans can hear are obviously at much lower frequency than those used in echolocation. Could we use the audible vocalizations as our dolphin version of a button response box? Perhaps. The interference of scanner sounds is an issue but again, with good acoustic engineering and careful tuning of the scanner peak frequencies I don't see why this couldn't be achieved, provided the vocalizations don't require movement of the head or body. These are issues that those doing human fMRI of audition and speech already deal with.

So long, and thanks for all the fish

Functional MRI of dolphins would seem to be a reasonable experiment to pursue if one has sufficient resources and the freedom to perform extensive customization of a scanner. I don't foresee any major technical impediments even if the logistics would be a tad involved. And, as they are the second most intelligent species on this planet, after the mice, I think it would be fascinating to see a dolphin's brain in action. Who knows, perhaps with fMRI we will be able to decode the dolphins' warnings of our imminent destruction by the Vogons. Or we could try to find a Babel fish to shove in an ear. Choices, choices.


1 comment:

  1. Some interesting perspectives on the various mental abilities of animals, including cetaceans: