Thursday, February 26, 2015

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.  
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. 

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