How Frogs Survive Being Frozen Solid

The wood frog is the only amphibian found north of the Arctic Circle where its body is wet and cold-blooded which represents an animal that almost certainly does not come to mind when thinking of the frost-covered tundra yet this amphibian defies the logic that applies to most other cold weather animals.

The wood frog does not survive in icy climates by resisting the cold but by embracing it where it freezes and thaws with the environment with up to 70 percent of its body water freezing with every cold snap representing a fundamentally different survival approach than heat generation or insulation.

When winter starts to take hold the wood frogs take shelter in the leaf litter of the forest floor where they assume a crouched position with the limbs drawn in close to the body and their head lowered which is called the water holding position and helps to reduce the amount of water lost to evaporation over what could be months of being frozen.

Ice formation in the wood frog begins in the extremities then spreads throughout the entire body cavity surrounding the abdominal organs where eventually as much as 70 percent of the frog’s body water is frozen and vital signs completely stop creating the appearance of a very dead frog with body frozen solid and eyes glazed over.

Normally ice formation like this should shred tissue where the crystals physically rip cells apart and freezing also normally wrecks cells by affecting the flow of fluids into and out of them because before ice forms inside cells it forms in the spaces between them which reduces the volume of liquid there increasing the concentration of dissolved salts and other ions causing water to rush out from the cells to compensate causing them to shrivel and die.

Frozen wood frogs have blood containing huge amounts of glucose at 400 millimolar concentration where this glucose acts as a protective molecule known as a cryoprotectant which are molecules such as alcohols or sugars that animals can use to lower water’s freezing temperature inside their bodies essentially acting as a biological antifreeze and if you tasted a frozen frog it would taste sweet.

To avoid the fate of ice damage the wood frog’s body carefully orchestrates the entire freezing process to avoid ice crystallization and dehydration in vital parts of the body where during the course of freezing they change over their metabolism, reorganize themselves, are not damaged by the physical ice, not damaged by the anoxia, and not damaged by the dehydration.

Once the frog’s body senses that the temperature is near freezing the frog’s liver begins to dump massive amounts of glucose into its bloodstream followed by glycerol and alcohol where more glucose starts to be packed into the frog cells representing a rapid and coordinated metabolic response to impending freezing conditions.

More glucose starts to be packed into the frog cells along with glycerol and alcohol to prevent ice from forming inside cells and to prevent any drastic loss in volume due to the dehydrating effects of ice formation where this intracellular cryoprotectant accumulation is critical for cellular survival during freezing.

At the same time water begins to move out of the vital organ cells and pools in the extracellular space where ice nucleating proteins trigger the ice to freeze in these specific places in sheets between the skin and muscle layers, in small spaces such as the lens of the eye or the ventricles of the brain, everywhere except the critical organs and cells.

In the big picture of history organ transplants are a relatively recent thing where it was not until 1954 that the first human organ transplant was successfully completed which was a kidney transplanted from one identical twin brother to the other and in 1967 the first liver and the first heart were transplanted marking the beginning of the era of organ transplants.

Wood frogs are adapted to being frozen where if you take a wood frog and you keep it in the lab and you do not freeze it, it does poorly because it just uses up all of its fuel including all of its muscle, all of its fat, all of its glycogen and it dies demonstrating surprisingly that they do not just withstand the cold but actually depend on it.

In the United States there are over a hundred thousand people on a waiting list to receive an organ transplant but only around forty thousand transplants are performed each year where seventeen people die per day waiting for an organ that never came and much of this is due to a challenging supply network constricted by the inherent lifespan of organs once they have been removed from the body where with the standard cooling methods a liver or pancreas can survive for 12 hours outside the body and a heart for only six.

Shortly after the first liver transplant researchers attempted the first frozen preservation of livers where using glycerol as a cryoprotectant they froze canine livers at negative 20 degrees Celsius for periods between 24 hours to 2 weeks but when the livers were then transplanted into healthy recipients the animals only survived for a few hours because the livers had suffered serious damage to their cells, most notably to their blood vessels.

Years later researchers tried again this time armed with more knowledge of freeze tolerant animals like the wood frog where mimicking how the wood frog undergoes freezing in nature researchers infused rat livers with glycerol and brought the temperature down to just negative three degrees Celsius and did so slowly mirroring what happens to the frogs in the wild and when these livers were transplanted back into the original donors they did produce bile and one animal even lived for five days but sadly none of the animals ultimately survived.

After decades of work in this field researchers are coming to the conclusion that ice is just too damaging to tissues even in the presence of cryoprotectants where the frogs are able to withstand such freezing due to an amalgamation of multiple adaptive strategies including anoxia tolerance, dehydration tolerance, metabolic rate suppression, huge amounts of cryoprotectants, and targeted ice nucleating proteins.

Researchers argue that perhaps looking at freeze tolerance was never the right place to look where if the goal of freezing is simply to reduce the cellular needs of organs maybe there is a way to achieve this without the freeze and this then raises the obvious question that if freezing temperatures are not required for metabolic rate suppression can we bypass the ice altogether and can we simply find a way to shut off the metabolism of organs to allow them to last longer.

The frogs are able to withstand such freezing due to an amalgamation of multiple adaptive strategies including anoxia tolerance, dehydration tolerance, metabolic rate suppression, huge amounts of cryoprotectants, and targeted ice nucleating proteins where we will struggle for a long time to recreate all of these elements working in perfect unison inside organs each with different sizes, metabolic requirements and cell types.

The gray mouse lemur is the particular animal model that researchers are currently researching where they are one of a few primate species that have recently been discovered to hibernate and they do so in warm weather since every hibernator you know of hibernates in the cold like bears, ground squirrels, bats, and mice but lemurs hibernate in Madagascar where it has never been cold in Madagascar for 60 million years because Madagascar has two sides being summer and winter but they are really wet and dry seasons.

When a lemur finally turns off its metabolism its body temperature goes down from 37 degrees Celsius down to 35 degrees Celsius which is not cold but warm where they turn off their metabolism with known molecular mechanisms because we study them including turning it off with microRNA, taking message RNAs and putting them in prisons by developing parts of their cells which are prisons for mRNA so they cannot make any proteins, doing it by phosphorylating and turning off metabolic pathways, reorganizing transcription and translation, and reaching into the nucleus and making certain DNA cannot be developed any further.