The Wood Frog is Leading the Way to Suspended Animation

By:  Patrick T. Paine L.Ac.

THIS IS THE STORY OF THE WOOD FROG, Lithobates sylvaticus. 

Winter is coming, the wood frog knows it, and fortunately is well prepared to handle it.  As the temperature in the Arctic Circle (his range is huge and extends down into southern wisconsin) drops to well below freezing he does something that was thought to be reserved for astronauts in science fiction novels, he enters a protected state of suspended animation.  His heart has stopped pumping and his tissues are frozen.  When the spring arrives and the sun melts the snow around him he begIns to thaw.  As he warms, his 3 chambered heart starts to beat, his eyes blink, his breath returns, and finally he moves his limbs.  Within a very short time he has returned to full activity level and can even begin mating.  

COLD DUDE!

There is much to admire in the wood frog but what stands out is the ability to freeze without his cells being destroyed by the damaging effects of ice.  This is a truly amazing.  What types of questions does your imagination dream up when presented with the re-animation of a frozen wood frog?  Here are mine with the quicky answer below it.

•  How does he do it?
• He uses glucose and urea to prevent the damaging effects of ice.
• When and why did this ability develop?
• Via convergent evolution, many species developed various anti-freeze proteins to survive 1-2 million years ago when the planet cooled and glaciated.
• Do other organisms have this same ability?
• There are a diverse array of anti-freeze proteins (AFPs) and anti-freeze compounds used by many organisms including the turtle, salamander, beetle, fish, and rye.  
• Could we apply this secret to ourselves and develop life saving cryo-surgerical techniques?
• AFPs have been used to successfuly freeze organs and reintroduce them into rats.
• Could our understanding and application of this ability lead to suspended animation for applications in medicine or even long range space travel?
• Potentially but there are many hurdles to cross before we get there.

Now before we examine this amazing ability and each of the questions above about the wood frog lets look at some of the basic characteristics that defines it as an organism.  It is an ectotherm and therefore relies on the environment for determining body temperature.

It's getting hot in here

Yo momma is cold blooded

Normally, being an ectotherm sounds like a disadvantage in cold environments compared to endotherms but in this case you would be wrong.  Because the wood frog and other amphibian ectotherms had to “solve” the problem of being cold or freezing they developed extra capabilities that endotherms lack.  In this case they developed anti-freeze compounds.

Another interesting physiological trait of the wood frog is that it has a three chamber heart.   

Within the sole ventricle of this heart it receives deoxygenated return flow from the body and oxygenated blood flow from the lungs.  These two mix and are pumped back into the circulation where it can feed tissues or pick up more oxygen from the skin or lungs.  This type of heart should not be confused with the three chamber heart in the turtle or snake which has a shunt to divert blood flow in post-prandial conditions.  Have a look below and notice the differences between the hearts of the ectotherms (frogs and turtles) and the endotherms (humans).  The honey bee is special because it is the only organism with a 5 chamber heart, unless you count the occasional birth abnormality found in humans.  

2 Loops but 3 chambers in the frog.  Only endotherms have 4 chambers

Only the bee has 5 chambers

Now that you are a little more familiar with the wood frog anatomy and physiology it still doesn’t really answer how do they withstand the cold.  First of all we need to understand why freezing kills us.  It is a slightly trickier answer than you would first expect.  My first thought was the physical and mechanical damage from the expansion of water when it form ice.  Well, this is only a little bit right.  It actually has to do with osmotic gradients (yes, osmoregulation is back to haunt you).  As the ice first forms in the interstitial space it forms a crystal structure.  The local solutes that were formerly dissolved within the now frozen water joins the remaining water and increases the osmolarity.  This increase in extracellular osmolarity means that the water in the cell will rush out along the concentration gradient.  The relative intracellular osmolarity jumps and becomes a deadly environment for the cell.  It is also undergoes a severe decrease in volume which can physically destroy the plasma membrane.  (Larson et al 2014) 

The ice crystal resembles little daggers

The key two points to remember about why ice kills are:  A jump in intracellular ion concentration and cell shrinkage due to water outflow and mechanical damage from extracellular ice crystal formation.  

If you were to guess how the anti-freeze proteins and compounds prevent cell and tissue damage what would you say?  Based on your new knowledge about the pathology of freezing you might think it blocks the change in intracellular ion concentration and it minimizes the spread and location of ice crystal formation.  Well, again, you would be partially right.  Let’s examine the process from the beginning and see what happens step by step.  

At first, the temps only drop below freezing at night and warms back up during the day, so the wood frog goes through around 15 freeze/thaw cycles.  This is a good thing.  Frogs that were frozen in the lab were not able to survive at the extremely cold temps and for the length of time that wild frogs could and it is because of this initial freeze/thaw cycle.  It allows for a build up of glucose over time.  The glucose in the cells comes from the breakdown of glycogen in the liver and it directed to the tissues and then the intracellular space.  This increase in intracellular osmolarity prevents the loss of water during freezing.  Another key aspect to prevent cellular damage is the strengthening of the plasma membrane by the AFPs.  (Lawson et al. 2005)

Not all AFPs work via the same mechanism.  Some arctic fish have AFPs that physically slow down the formation of bonds between water molecules as they form ice crystals.  

This is all fascinating but what about applications.  Can we use any of this knowledge?  It ends up we can but only a little.  At this point the successful cryoprotection with AFPs have allowed the freezing, thawing, and transplantation of rat and pig hearts (Amir et al. 2005, Banker et al. 1992).  This could be a huge advance for human transplantation which is only able to cool the organs and not freeze them.  A bank of frozen organs for transplant would be a massive step forward.  Of course we can already freeze sperm, eggs, and embryos but the major hurdle at this point is the larger tissue and organs.  This is compounded by the fact that the AFPs are toxic at the doses that a human would require.  There might be some hope with kidney storage using vitrification but it still needs work (Fahy et al. 2009). So, if some of you are hoping, like Ted Williams, to be frozen indefinitely in a state of suspended animation until you can be safely thawed we aren’t quite there yet.  On a side note, I’m curious, what’s the difference between death and suspended animation?  By any definition they look the same on paper but I’ll leave that as a topic for another day.

Puny endotherms.  Let's see you freeze solid. 

About the author:

Patrick Paine L.Ac. has found that the lyrics to The Gambler make more sense as you get older. 

Literature Cited:

Amir, G., Rubinsky, B., Basheer, S.Y., Horowitz, L., Jonathan, L.,  Feinberg, M.S., Smolinsky, A.K., and J. Lavee. 2005. Improved viability and reduced apoptosis in sub-zero 21-hour preservation of transplanted rat hearts using anti-freeze proteins. Journal of Heart and Lung Transplant. 24:1915-29.

Wang, T., Banker, M.C., Claydon, M., Hicks, G.L., Layne J.R. 1992. Freezing preservation of the mammalian heart explant. III. Tissue dehydration and cryoprotection by polyethylene glycol,” Journal of Heart and Lung Transplant. 11:619-23,

Layne, J. R., Lee, R. E., Jr and Huang, J. L. 1990. Inoculation triggers freezing at high subzero temperatures in a freeze tolerant frog (Rana sylvatica) and insect (Eurosta solidaginis). Can. J. Zool. 68, 506-510.

Storey, K. B. and Storey, J. M. 1996. Natural freezing survival in animals. Annu. Rev. Ecol. Syst. 27, 365-386.

D. J. Larson, L. Middle, H. Vu, W. Zhang, A. S. Serianni, J. Duman, B. M. Barnes. 2014. Wood frog adaptations to overwintering in Alaska: new limits to freezing tolerance. Journal of Experimental Biology.  217: 2193 

Fahy GM, Wowk B, Pagotan R, Chang A, Phan J, Thomson B, Phan L. 2009. Physical and biologcial aspects of renal vitrification. Organogenesis 5:167–175. 

Newest sunscreen product on the market: hippo sweat

By Evelyn Puspitasari

We have less than a month before spring hits, then soon after summer will be here. We're also  located in Central Coast. You know what that means? THE BEACHES.

Stock up on sunscreen everyone. We're hitting the beach.

We all know that forgetting to put sunscreen on may result in sunburn. Most of us have unfortunately experienced the painful reddish burn on our skin due to prolong exposure to the sun. Sunburn is actually a burn to living tissue such as our skin tissue by overexposure to ultraviolet radiation, commonly from the sun's rays. If you're lucky, less exposure to UV radiation will produce suntan instead.

Okay, sunscreen is good. Got it. But is it really? Turns out that well... sunscreen sort of has bad side effects... Autier et al. (2007) suggests that increase sunscreen usage in individuals may increase risk of cutaneous melanoma because people feel it is okay to be out in the sun for longer period of time. Some people also believed that applying sunscreen could reduce risk to skin cancer, unfortunately this is not true either. Hence, there is a lot of research going on to find a safer alternative to our commonly used sunscreen.

Well. I propose to use hippo sweat as sunscreen.

Yes, hippopotami are cute beasts. The common hippopotamus, Hippopotamus amphibious L. is the third largest and heaviest mammal after elephants and white rhinos. They are widespread across lakes and rivers throughout Africa, although you can probably go to your local zoo and find hippos there too. They are herbivores, but they are also vicious. Water is actually required for their thermoregulation, which is why hippos are known amphibious and always found near water. They have unique skin with thin epidermis and no sweat glands. Out of water, they lose water much more quickly than other mammals, which is why they spend a lot of time in the water to keep their bodies cool since they don't sweat.

Although they don't have sebaceous glands, they do have mucus-secreting glands that produce "sweat". Hippos' subdermal glands are the ones responsible to produce the colorless viscous "sweat" that will cover their entire hairless body and keep their skin moist. Within a few minutes, the colorless "sweat" will turn red, then gradually turning brown after a few hours because of pigment polymerization. This would explain the story of "Hippos secrete a blood-like sweat" that was spread by ancient travelers all over the world.

A study done in Japan by Saikawa et al. (2004, 2007) with the help of animal keepers at the Ueno Zoological Gardens and the Kyoto Municipal Zoo succeeded in isolating hippos sweat! The Saikawa team bravely wiped hippos bodies with paper towels to get the "sweat" secretion, so that pigment purification could be done.

They managed to isolate two pigments - a red and an orange pigment. They dubbed the red one as hipposudoric acid, whereas the orange one is called norhipposudoric acid. These pigments are apparently stabilized to some extent by interacting with the highly alkaline (pH 8.5-10) hippo "sweat", altough overtime will still turn into a brown polymeric material.

Knowing the behaviors of hippos, they almost always have wounds and scratches due to battles between one another or with predators. Is it possible that these pigments have antibacterial properties? The Saikawa team asked the same question and proceeded to grow bacteria in the presence of the red and orange pigments. Turns out both pigments are effective at inhibiting growth of some Gram-negative bacteria such as Pseudomonas aeruginosa and Klebsiella pnemoniae, although the red pigment has a stronger antibiotic effect than the orange one.

Wait, they are pigments! Think! Wavelength! Absorbance! Apparently the pigments absorb light in the UV-visible range (200-600 nm) indicating that the pigments may behave like sunscreen. The researchers conclude that hippo "sweat" not only cools the body, but also acts as sunscreen and protects the hippos against bacterial infection. Win win!

So why don't we have this on the market yet, you ask? Well remember how I said that hippos were vicious beasts? Yeah.... Getting "sweat" samples from hippos is not the easiest thing to do, which means producing a synthetic version of their "sweat" will take longer than expected. If you'd like to witness how scary a hippo can be, feel free to watch this R-rated video below:

Are you terrified now? Here. I'm sorry. This is my peace offering. Click it, you'll feel better afterwards, I promise.


References:
Berwick, M. 2011. The good, the bad, and the ugly of sunscreens. Clinical Pharmacology & Therapeutics 89:31-33.
Donawho, C. and P. Wolf. 1996. Sunburn, sunscreen, and melanoma. Current opinion in oncology 8:159-166.
Hashimoto, K, Y. Saikawa, and M. Nakata. 2007. Studies on the red sweat of the Hippopotamus amphibius. Pure and Applied Chemistry 79:507-517.
Saikawa, Y., K. Hashimoto, M. Nakata, M. Yoshihara, K. Nagai, M. Ida, and T. Komiya. 2005. Pigment chemistry: the red sweat of the hippopotamus. Nature 429:363-363.
Timbuka, C.D. 2012. The ecology and behaviour of the common hippopotamus, Hippopotamus amphibious L. in Katavi National Park, Tanzania: Responses to varying water resources: University of East Anglia.

Image and video references:
http://giphy.com/gifs/beach-water-ocean-f45816CTsKd7G
http://giphy.com/gifs/friends-black-sun-smMHbiEP5jyla
http://giphy.com/gifs/cute-hippo-yRtpTJ2Sz3Yvs
https://www.youtube.com/watch?v=c5Pmd515u5U&spfreload=10
http://cen.acs.org/content/cen/articles/82/i22/HIPPOS-SWEAT-PROTECTION/_jcr_content/articlebody/subpar/articlemedia_1.img.gif/1375723112826.gif
https://www.youtube.com/watch?v=WfrG95GyU9U

A Beautiful Mind

by Naiyerah Kolkailah

When we’re sleep deprived, it’s not just outward beauty that gets compromised.

Our brains also take a big hit. We have trouble focusing, processing information easily, and remembering things. Sleep deprivation has actually been shown to negatively affect learning, memory, and cause seizures. It can even be fatal in extreme cases. Rodents and flies that didn’t get enough sleep died within days or weeks. Also, people who have insomnia can eventually get dementia if the condition is untreated, and they can die within months or years.

We know sleeping is vital, but the question of why sleep serves a restorative function for our minds has been a mystery for decades. One of the most recent findings on sleep is related to the process of waste clearance in the brain.

In our bodies, waste clearance takes place through the lymphatic system. The lymphatic system is a network of vessels that runs parallel to the cardiovascular system. While blood vessels deliver oxygen and nutrients to the tissues, the lymphatic system collects excess proteins and metabolic wastes that accumulate in the interstitial fluid (between cells) in tissues--and dumps them into the blood for degradation in the liver.

Take a look at the lymphatic system in the image above. You’ll notice that there are no lymphatic vessels in the brain. Unlike blood vessels that form a network within the entire brain volume, lymphatic vessels aren’t even present! The brain is a very metabolically active organ that produces large amounts of metabolic waste. Neurons are also very fragile and sensitive to toxic waste products. Some waste products can hinder synaptic transmissions or cause irreversible injury to neurons. With so much at stake, metabolic waste needs to be cleared efficiently so neurons can function properly. How does the brain manage without a lymphatic system? The answer lies in a unique waste clearance system reserved especially for the brain.

In the brain, there is a clean fluid known as cerebral spinal fluid (CSF). That fluid surrounds the brain, and receives waste products from inside the brain for eventual disposal. How do the metabolic wastes make their way out to the CSF? Instead of using another set of vessels in a tight space packed full of cells, the CSF itself gushes through the brain and washes out the interstitial waste products on the outside of the arteries.

Check out these images from a time lapse video of a living mouse injected with fluorescent CSF tracers.

The left frame shows blood vessels on the brain surface, and the right frame shows the blood vessels inside the brain.

Within a few moments, you can see CSF (fluorescent green) rushing in and coating all the blood vessels! The CSF starts appearing at the brain surface, and then makes its way into the brain.

All the blood vessels are coated within seconds! 

Now, are you wondering where sleep comes in? Ok, here it comes. The researchers who conducted this study (Xie et al.) showed that the influx of CSF that clears waste from the brain doesn’t just happen at any time. It only happens during sleep!

Here is a graph (left) showing CSF influx over a period of 30 minutes. The influx is significantly higher in mice that are awake (green) than mice that are sleeping (orange). The graph on the right compares the intensity of the tracer between the two states (sleep and awake). The graph shows that CSF intensity is significantly higher when the mice are sleeping than when they're awake.

The images below are from a time-lapse video showing the blood vessels (blue) in the brain of an awake mouse (left frame), and the blood vessels in the mouse when it sleeps (right frame). 

The CSF appears in red and coats the blood vessels, but only after the mouse falls asleep.

The researchers wanted to test if the sleep-wake cycle is what induces the CSF influx, so they conducted a similar experiment on anesthetized mice. They used Ketamine/xylazine (KX) to anesthetize the mice, and they found very similar results as in the natural sleep experiment.

The graph on the left above shows the CSF tracer increasing over time in the anesthetized state but not the awake state. The graph on the right shows that the intensity of the CSF is significantly higher in the anesthetized state than in the awake state.

The results of this second experiment show that it is the sleeping state itself (whether natural or anesthetically induced) that allows CSF influx into the brain. In another part of this study, the researchers found that during sleep, the interstitial space volume expands by over 60%. It is that expansion that allows CSF to effectively flow through the brain and clear out metabolic waste.

One of the most dangerous metabolic waste products that get cleared from the brain during sleep is Amyloid beta (Aβ). Aβ is a protein that accumulates in the brain when we’re awake, and it is linked to neurodegenerative diseases. Clinical studies have shown that Alzheimer’s patients who didn’t sleep well or didn’t get enough sleep had a buildup of Aβ in their brains. These studies show that the brain’s inability to clear out waste can contribute to the development of Alzheimer’s and similar neurodegenerative diseases.

Xie et al. wanted to compare the clearance rate of Aβ from the brain during sleep and during wakefulness, and they found that Aβ is cleared most efficiently during sleep.

The left graph above shows a time-disappearance curve of radiolabeled Aβ (I-Aβ) in three groups of mice: awake (orange), asleep (green) or anesthetized with KX (red). The curves show I-Aβ clearing at a faster rate in the sleeping and anesthetized mice than in the mice that are awake. The bar graph on the right gives the rate constant of Aβ clearance from the clearance curves. Aβ cleared twofold faster in the sleeping mice than in the awake mice, and there was no significant difference between the clearance rate in the sleeping and the anesthetized mice.

Figuratively, we know that sleep helps clear the mind. Now, this study shows how that clearance happens physiologically. Sleep is very precious! It’s essential. Make sure you get quality sleep to keep that beautiful mind of yours. Now, I’ll take my own advice so I don’t end up doing this.

References:

Illif, J. One more reason to get a good night’s sleep (Video). Accessed from web Feb. 24, 2015. <http://www.ted.com/talks/jeff_iliff_one_more_reason_to_get_a_good_night_s_sleep?language=en#t-443069>

McKinley, J., A. McCarthy, and T. Lynch. 2013. Don’t lose sleep over neurodegeneration-it helps clear amyloid beta. Front Neurology 4: 206

Stickgold, R. 2006. Neuroscience: a memory boost while you sleep. Nature 444: 559-60.

Xie, L., H. Kang, Q. Xu, M. Chen, Y. Liao, m. Thiyagarajan, J. O’Donnell, D. Christensen, C. Nicholson, J.J. Iliff, T. Takano, R. Deane, M. Nedergaard. 2013. Sleep Drives Metabolite Clearance from the Adult Brain. Science 342: 373-377.

Images:

Cardiovascular & Lymphatic Systems: http://legacy.owensboro.kctcs.edu/gcaplan/anat/Notes/API%20NOtes%20A%20Body%20Systems%20and%20Cavities.htm

Sleep Meme:

http://icanhas.cheezburger.com/tag/bad-sleep

http://funny-pictures.picphotos.net/memes-about-sleep-meme-comics-picture/

Jeff Illif’s TED Talk: http://www.ted.com/talks/jeff_iliff_one_more_reason_to_get_a_good_night_s_sleep?language=en#t-443069