Hepatocyte responses to in vitro freezing and β‐adrenergic stimulation: Insights into the extreme freeze tolerance of subarctic Rana sylvatica

Abstract

The wood frog, Rana sylvatica LeConte 1825, is a freeze-tolerant amphibian widely distributed in North America. Subarctic populations of this species can survive experimental freezing to temperatures below -16 °C, whereas temperate populations tolerate freezing only at temperatures above -6 °C. We investigated whether hepatocytes isolated from frogs indigenous to Interior Alaska (subarctic) or southern Ohio (temperate) had distinct characteristics that could contribute to this variation in freeze tolerance capacity. Following in vitro freezing, cell damage, as assessed from lactate dehydrogenase leakage, was similar between samples from Alaskan and Ohioan frogs. Preincubation of cells in media containing glucose or urea, the two primary cryoprotectants used by R. sylvatica, markedly reduced freezing damage to hepatocytes; however, results suggested that cells of the northern phenotype were comparatively more amenable to cryoprotection by urea. Stimulation of isolated hepatocytes with β-adrenergic agonists, which simulates the freezing-induced cryoprotectant mobilization response, gave rates of glucose production from endogenous glycogen reserves that were similar between the populations. Our findings suggest that extreme freeze tolerance in subarctic R. sylvatica does not require an enhanced ability of the liver to resist freezing stress or rapidly mobilize cryoprotectant.

Effect of Ice Nucleation and Cryoprotectants during High Subzero-Preservation in Endothelialized Microchannels

“…Instead, new cryopreservation methods should look to nature for inspiration. Various species of vertebrates, such as Rana sylvatica (the wood frog), achieve a thermodynamically stable, noninjurious frozen state at high subzero storage temperatures ranging from −6 to −22 °C. − Using cryomicroscopic analysis, researchers have detailed the mechanisms of ice propagation utilized by freeze-tolerant wood frogs in nature . In this study, micrographs of liver slices showed continuous ice formed along the vasculature with the parenchyma remaining ice-free and considerably shrunken because of cellular dehydration.…”

“…Various species of vertebrates, such as Rana sylvatica (the wood frog), exhibits the ability to survive long periods of time in a partially frozen state with the whole animal, including every single organ demonstrating the ability to live in the presence of extracellular ice without injury . It has been shown that subarctic populations of wood frogs can tolerate temperatures close to −18 °C, although temperature ranges can vary between −5 and −22 °C. − …”

New Approaches to Cryopreservation of Cells, Tissues, and Organs

“…Notwithstanding the strategy adopted by the arctic wood frog for high-subzero survival in a partially frozen state, there is extensive evidence that mammalian organ banking might be better served by freeze avoidance rather than freeze tolerance [108, 112], which is also employed in nature. A third option is to leverage the strengths of these two approaches for nonfrozen subzero storage in thermodynamic equilibrium.…”

“…One good example from the at least 45 animals that can survive long periods of time at high-subzero temperatures in a state of “suspended animation” is the wood frog ( Rana sylvatica ), surviving with 65–70% of the total body water as extracellular ice [112]. One of the most critical strategies for freeze tolerance involves the synthesis of high amounts of low-molecular-weight carbohydrates (glucose in wood frogs), which provide colligative resistance to detrimental decreases in cell volume while also serving to stabilize the phospholipid bilayer of membranes and to restrict the formation of intracellular ice [112-114].…”

“…One of the most critical strategies for freeze tolerance involves the synthesis of high amounts of low-molecular-weight carbohydrates (glucose in wood frogs), which provide colligative resistance to detrimental decreases in cell volume while also serving to stabilize the phospholipid bilayer of membranes and to restrict the formation of intracellular ice [112-114]. Importantly, research has shown that simple augmentation strategies such as increasing endogenous levels of cryoprotectants (via injection) prior to freezing have the capacity to improve survival and extend the time in a frozen state in a certain context from 2 weeks to 49 days [113].…”