A third mechanism of damage relates to the denaturation of proteins as a function of both temperature and change in the intracellular ionic content. Most cells and tissues can withstand brief cooling to above freezing temperatures, in the time scale typical of a cryosurgical procedure and under the cooling circumstances typical of cryosurgery.
Major exceptions are cells that are highly sensitive to their ionic content, such as platelets. Cooling platelets to temperatures lower than their lipid phase transition temperature allows calcium influx, which appears to trigger platelet activation. This could lead to a cascade of events that would end in platelet aggregation and the eventual obstruction of blood vessels in the cooled region around the frozen lesions. Other cells whose function is strongly dependent on their ionic content are muscle cells, in particular in the heart and around arteries. These may be also damaged in the cooled region beyond the frozen lesion.
The effect of freezing
The thermal processes during freezing for preservation (cryopreservation) are different from the thermal processes during cryosurgery.
In cryopreservation cells and tissues are frozen in vitro, they are usually frozen with uniform conditions to very low cryogenic temperatures, are kept in a frozen state for long periods of time and most important are frozen in the presence of chemical additives that improve survival.
In contrast, in cryosurgery the tissue is frozen in vivo, it experiences a large variation in cooling and warming conditions and in a frozen state it experiences a wide range of temperature, from the phase transition temperature on the outer edge of the frozen lesion to cryogenic temperatures near the probe.
More relevant to understanding the mechanism of damage during cryosurgery are experimental results of cancer cells frozen with specified cooling rates to different subzero temperatures. For cooling rates of 1 and 5oC/min there is a gradual, almost linear increase in cell death to temperatures of about -40oC. For higher cooling rates of about 25 oC/min there appears a sudden step like increase in cell death at a temperature of about -10oC. At the beginning of the freezing process cells accumulate on the change of phase interface, which has the appearance of a vertical line. The increased solute concentration on the change of phase interface has the effect of colligatively lowering the temperature of the change of phase interface. Because thermal diffusion is much faster than mass diffusion the increased concentration and related change in phase transformation temperature leads to a phenomenon known as "constitutional supercooling" and the so-called Mullins-Sekerka interface instability. It causes the planar freezing interface to become unstable and take the finger like shape. In this configuration the concentration of the solution at the tip of the finger like ice crystal structure is very close to the bulk solution concentration and the rejected solutes become accumulated between the fingers like ice crystal structures. The cells in the freezing solution are unfrozen and find themselves in the high concentration solute channels, between the ice crystals. This is the hallmark of the process of freezing in biological materials. Although referred to as freezing of tissue or cells, in fact during most of the freezing processes the freezing begins in the extracellular milieu and the interior of the cell is unfrozen.