"What's So Hot About Cryosurgery?" TUMOR TIDBITS, A BIWEEKLY EMAIL NEWSLETTER FROM GULF COAST VETERINARY ONCOLOGY Number 93; March 21, 2003. ======================================================================= THIS WEEK'S TUMOR TIDBIT: What's So Hot About Cryosurgery? ======================================================================= Cryosurgery is a surgical technique that employs freezing to destroy undesirable tissue. Developed first in the middle of the 19th century it has recently incorporated new technologies and is becoming a fast growing minimally invasive surgical technique. This somewhat lengthy Tumor Tidbit reviews the biochemical and biophysical mechanisms of tissue destruction during cryosurgery. History of cryosurgery Cryosurgery, sometimes referred to as cryotherapy or cryoablation, is a surgical technique in which freezing is used to destroy undesirable tissues. The prefix "cryo" (from the Greek word "kruos" for cold) refers to temperatures below 120 K; cryosurgery deals with temperatures below the freezing temperature of tissue, i.e. about 273 K. In cryosurgery the freezing probe or cryogen are applied to a particular tissue site, and the freezing domain propagates outwards from the site of application into the tissue. Therefore, the extent of tissue affected by the treatment is much greater than the tissue in contact with the cryogen or the probe. As such, cryosurgery is probably the first minimally invasive surgical technique. The cryosurgical probes developed in the 1960's allow for precise application of cryosurgical treatment deep in the body. This unique ability made cryosurgery very promising and resulted in the expansion of the method during this era. However, the minimally invasive nature of cryosurgery leads to difficulties with controlling the procedure. First, because the freezing propagates from a probe or a cryogen outward, the extent of the tissue affected by freezing cannot be determined visually by the surgeon, unlike the more conventional surgical resection techniques. Second, indiscriminant freezing itself does not necessarily destroy the tissue, a topic that will be discussed in the next section. Therefore, while the new cryosurgical probes could be applied at a precise location, their effect on the tissue treated by freezing was not precise. This lack of precision was soon recognized and lead to a reappraisal and eventual decrease in the use of the method during the 1970’s. Cryosurgery lost its popularity even for surgical applications in which it had a proven beneficial effect. Laser techniques began to replace cryosurgery as a new technology for destruction of undesirable tissues. By the early 1980’s, the field of cryosurgery had essentially reverted back to the original application in which it was traditionally employed: dermatology. The mechanisms of tissue injury during cryosurgery. To apply cryosurgery precisely, it is imperative to know the following: a) what are the mechanisms of tissue destruction during cryosurgery and b), how to evaluate the extent of tissue freezing and the thermal history in the frozen lesion. In cryosurgery tissue is frozen with a cryosurgical probes that is brought in good thermal contact with the undesirable tissue. Usually, the probe is cooled through the internal circulation of a cooling fluid. The cooling fluid gradually extracts heat from the tissue, through the probe. Within several minutes after cooling begins, the temperature of the tissue in contact with the probe reaches the phase transition temperature and the tissue begins to freeze. As more heat is extracted the temperature of the probe continues to drop and the freezing interface begins to propagate outward from the probe into the tissue. A variable temperature distribution in both the frozen and unfrozen regions of the tissue ensues. The freezing interface propagates outward until either the flow of the cooling fluid is stopped or until the heat that comes from the live tissue surrounding the frozen lesion becomes equal to the amount of heat that the cooling fluid in the cryosurgical probe can remove. At that time the frozen tissue has a temperature distribution that ranges from a low cryogenic temperature at the tissue surface in contact with the probe to the phase transformation temperature on the outer edge of the frozen lesion. The unfrozen tissue surrounding the frozen lesion has a temperature distribution from the phase transformation temperature at the outer range of the frozen lesion to the normal body temperature. In typical cryosurgical protocols, after freezing was completed the cooling system keeps the tissue frozen for a desired period of time, followed by heating and thawing. The primary mechanism for heating the frozen tissue is from the blood circulation and metabolism of the surrounding tissue. Sometimes the frozen tissue is also warmed from the probe surface by a warming fluid circulating through the cryosurgical probe. Depending on the desired outcome the tissue is frozen again, after complete thawing, or after partial thawing. The cryosurgical procedure can be performed with several cryosurgical probes to generate a particular shape of the frozen tissue, which corresponds to the shape of the undesirable tissue. A typical cryosurgery procedure lasts between several minutes and an hour. It seems that every cell of the tissue may experience a different thermal history. The cells near the cryosurgical probe surface will be cooled with a higher cooling rate and to lower temperatures than those farther away from the probe. The cells at different locations in the frozen lesion will be at different temperature for various periods of times, as a function of their distance from the probe surface, the cooling fluid employed, the shape of the cryosurgical probes, the number of the cryosurgical probes used, the type of tissue frozen. Cell damage during cooling and freezing occurs at several length scales: nanoscale (Armstrong) - molecular, mesoscale (micron) - cellular and macroscale (millimeter) - whole tissue. The time scales relevant to cryosurgery range between single minutes to tens of minutes. The thermal regime can be also divided in the temperature range from body temperature to the change of phase temperature for the body physiological solution and the temperature range below the change of phase temperature. The damage during cryosurgery is of two types, acute - immediately during cryosurgery and long term. The effect of cooling Most types of mammalian cells and tissues can withstand low, non-freezing temperatures for short periods of time. The phenomena related to cooling occur primarily at the nanoscale, with typical consequences at the mesoscale. Cells are entities with a highly specific intracellular chemical content, separated from the non-specific extracellular solution by the cell membrane. The cell membrane acts as a selective barrier between the intracellular and the extracellular milieu. The membrane selectively controls the transport of chemical species into and out of the cell. Therefore the membrane must be mostly impermeable except at particular sites where it can control the mass transfer. The by-layer lipid structure of the cell membrane makes it impermeable. The mass transfer through the cell membrane is controlled through membrane proteins that span the membrane. Mammalian cells have become optimized to function at the temperature in which the organism lives. One aspect of cooling the cell to temperatures lower than their normal physiological temperature is the lipid phase transition process. The lipid membrane bilayer is in a fluid state during normal life temperatures. At lower temperatures and lower thermodynamic free energy the lipids undergo phase transition into a gel phase or into other three- dimensional structures with lower free energy. During the process membrane proteins become segregated and defects form between the proteins and the membrane bilayer. This phase transition process makes the cell membrane more permeable and allows usually ions to enter the cell in an uncontrolled way. Normally the membrane proteins control the intracellular composition by selectively introducing and removing ionic species from the cell interior. However life processes are temperature dependent chemical reactions. Lowering the temperature also reduces the efficiency of the membrane proteins and their ability to control the intracellular content. Therefore, during cooling, the intracellular composition and in particular the intracellular ionic content begins to change as undesirable ions diffuse into the cells and are not removed. The damage is cumulative, a function of time, and is particularly expressed when the cells are returned to their normal physiological temperature. Additional mechanisms of damage relate to the cytoskeleton. The cytoskeleton structure depends on chemical bonds between membrane proteins and the cell scaffold. Lowering the temperature weakens these bonds and makes them particularly vulnerable to mechanical damage. 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. Therefore it is not anticipated that tissues in areas around the frozen region would be severely damaged by the cooling they experience in the time scale of a cryosurgical procedure. 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 In cryosurgery, when 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. The freezing process begins in the extracellular milieu and the interior of the cell is unfrozen. At low temperatures, the extracellular concentration increases as cells shrink. This shrinkage is caused by the fact that the unfrozen cells are supercooled relative to the extracellular solution, which is in thermodynamic equilibrium with the ice. To equilibrate the difference in chemical potential between the extracellular and intracellular solutions, water will leave the cell through the cell membrane that is readily permeable to water. This causes an increase in the intracellular solute concentration, with a decrease in temperature. Increased hypertonic extracellular solutions damage the cells. The mechanisms are not entirely clear and they could relate to chemical damage or osmolality induced changes in the cell structure. There are several additional phenomena worth mentioning in relation to the hypertonic mode of damage. The damage produced by exposure to hypertonic solutions is rapid and seems to be independent of temperature and time of exposure. Experiments have shown that the percentage of death cells after freezing is larger than the percentage of death cells after exposure to a similar extracellular hypertonic solution. This suggests that mechanical interaction between ice and cells may contribute to cell death. This is a reasonable assumption, since ice rejects cells in the space between ice crystals. This may generate a mechanical force on the cells, whose cellular cytoskeleton is weakened by cold, and destroy them. Another possible mode of damage is the contact and interaction between ice and the lipid bilayer, which by itself may be damaging. Ice appears to usually form in the vasculature and propagate in the general direction of temperature gradients, but in and along blood vessels and glandular ducts. Therefore cells in tissue will probably experience both qualitatively and quantitatively similar mechanisms of hypertonic solution damage and intracellular ice formation damage like cells frozen in cellular suspensions. However, the analysis suggests that in tissue the dehydration of cells will most likely result in a disruption of the vasculature and of the connective tissues. Thawing and warming Thawing and warming has been studied much less than freezing. However, they can also induce cellular damage. 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. 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 immediately after thawing there is edema on the outer margin of the previously frozen lesion. Shortly thereafter the endothelial cells in the previously frozen region appear damaged, probably by the mechanism of blood vessel expansion during freezing, discussed earlier. 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 angiogenesis 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 thawing, 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. In addition to the verified mechanisms of tissue damage during cryosurgery there is anecdotal evidence that cryosurgery may result in a beneficial systemic immunological response. There is no doubt that a normal immune response exists in response to the tissue injury which freezing produces. However the usefulness of this immune response in treating metastatic tumors is not certain. Summary of tissue damage The thermal history during cryosurgery is complex and so is the mechanism of damage. Cooling rates vary throughout the frozen lesion from uncontrollable high near the probe surface to low near the outer edge of the frozen lesion. Temperatures range from cryogenic near the probe to body temperature. This complex thermal history combined with the complex mechanism of damage during freezing makes it difficult to predict the outcome of a cryosurgery protocol and the relation between the extent of freezing and the extent of tissue damage. Summary Cryosurgery is an important minimally invasive surgical technique. It can be potentially applied to any procedure in which scalpels are used to remove undesirable tissues. Currently cryosurgery is being used in many veterinary disciplines such as dermatology, urology, neurology, pulmonary medicine, cardiology, oncology and many others. With new applications comes the need for better cryosurgical probes. New cryosurgical systems using supercooled liquid nitrogen, Joule-Thomson refrigeration with gas mixtures, closed cycle Stirling refrigeration and heat pipe cooling have been all developed in recent years. To improve cryosurgery further, there is the need to develop a better fundamental understanding of the mechanisms of tissue damage during cryosurgery, to develop improved imaging techniques for cryosurgery, new and improved cryosurgical device technology and mathematical cryosurgery optimization techniques..... and prospectively designed, well controlled clinical trials. ======================================================================= As always, we hope this info helps and don't hesitate to call or email us Gulf Coast Veterinary Oncology! Kevin A. Hahn, DVM, PhD, Diplomate ACVIM (Oncology), drhahn@gcvs.com Janet K. Carreras, VMD, Diplomate ACVIM (Oncology), drcarreras@gcvs.com Glen K. King, DVM, MS, Diplomate ACVR (Radiology & Radiation Therapy), drking@gcvs.com Gulf Coast Veterinary Diagnostic Imaging & Oncology 1111 West Loop South, Suite 150, Houston, TX 77027 P: 713.693.1166 F: 713.693.1167 W: www.gcvs.com ======================================================================= Copyright © 2003, Gulf Coast Veterinary Oncology