Thawing and warming can also induce cellular damage. During warming, in a frozen state, ice has a tendency to recrystalize at high subzero temperatures, to minimize the Gibbs free energy. Recrystalization will cause further disruption of the extracellular space and may disrupt the macroscopic structure of the tissue. During thawing, as ice melts the extracellular solution can be briefly and locally hypotonic causing water to enter some cells and expand them and rupture the membrane. When the thawing is rapid some cells may remain hypertonic at body temperature, which could induce metabolic disruption and additional damage.
Thermal parameters specific to cryosurgery
It is common in cryosurgery to employ double freeze thaw cycles. Comparison with a single freeze thaw cycle shows that the second freeze thaw cycle will increase damage. Double and even triple freeze thaw cycles are now commonly used in cryosurgery. The mechanisms of damage during multiple cycles are most likely related to cell membrane damage during the hypertonic variations that the cells experience upon freezing and thawing and with temperature variation.
Damage to the vascular system is probably one of the most important macroscopic mechanisms of tissue damage in cryosurgery. During cryosurgery the frozen region is obviously occluded from the blood circulation. Experiments show that after thawing there is edema on the outer margin of the previously frozen lesion immediately. Shortly thereafter the endothelial cells in the previously frozen region appear damaged, probably by the mechanism of blood vessel expansion during freezing. Within a period of several hours after thawing the endothelial cells become detached, with increased permeability of the capillary wall, platelets aggregation and blood flow stagnation. Many small blood vessels are completely occluded within a few hours after cryosurgery. The loss of blood flow will ultimately result in ischemia and tissue death. It is thought that this mechanism of tissue destruction explains why cells appear to have succumbed to cryosurgery even in those areas in which the freezing parameters would normally not cause cell death. Cryosurgery is probably the first surgical technique that has used angionesis to treat cancer.
While most of the studies on the process of cell death during freezing have employed viability tests that evaluated survival of cells immediately after freezing and hawing, it appears that some cooling and freezing conditions may produce less lethal modes of damage, which eventually result in gene regulated cell death (apoptosis). Apoptosis can be triggered by a variety of conditions present during cryosurgery, such as hyperosmolality. Apoptosis will take place after cryosurgery was finished and can produce further cell death.