ReviewExperimental cryosurgery investigations in vivo☆
Introduction
Cryosurgery is the use of freezing temperature to elicit a specific reactive response in tissue. The nature of the response depends upon the severity of the freezing injury. Minor freezing injury causes an inflammatory response, but severe freezing kills cells and results in destruction of tissue. An understanding of the mechanisms of cell and tissue injury due to freezing is only about sixty years in development. Though experience with cold injury and therapy is described in ancient manuscripts and in experiments with frostbite in the 19th century, most current knowledge is based on investigations that began in the 1940’s, initially with frostbite and then with the use of freezing techniques to preserve cells or produce local lesions for physiological investigations. By early in the 1960’s, experimental studies had shown that cell and tissue death from freezing was due to direct cell injury and to the vascular stasis that developed shortly after the thawing period. The relative importance of these two mechanisms of injury was a matter for debate. Investigators of frostbite were impressed by vascular stasis produced by freezing, but investigators of cryopreservation focused on ice crystal formulation and its deleterious effects. Nevertheless clearly both mechanisms are operative in freezing injury of any type or cause.
Experiments pertinent to cryosurgery on local tissue injury by freezing, which also began about sixty years ago, produce lesions which resemble frostbite. Investigations in vitro and in vivo have shown that the major mechanisms of injury are due to direct cell damage, which begins as tissues falls into hypothermia, and the failure of the microcirculation which develops after thawing [60]. The considerable research, which has been done in vitro has provided detailed information on the effects of freezing on cells. But substantial differences may exist between the effects of freezing cells in vitro versus freezing in vivo. The in vitro work, performed in the absence of the vascular factor of injury, permits close examination of the effect of the diverse facets of the freezing and thawing cycle. Whether directed at cell preservation or destruction, the in vitro work is of substantial importance and recently has clarified the molecular-based mechanisms in the complex cell death cascade [11]. Nevertheless, in cryosurgery, the effect of freezing on the vasculature is clearly a critical and perhaps dominant cause of cell death. With thawing, the microcirculatory failure in the previously frozen tissue is progressive, resulting in vascular stasis in about 1 h, which insures cell death from ischemia. Therefore research in vivo by cryosurgical techniques is critical to knowledge of freezing for therapy.
The range of investigations in vivo is substantial and covers every facet of cryosurgery. Many experiments focus on the technical features of cryosurgery, that is, the manipulation of the freeze–thaw cycle to enhance destruction or to achieve a cell-selective response. Other work investigates the monitoring of the freezing process in the tissue, principally by imaging, but also by other techniques. Many investigations detail the effects of freezing tissue in terms of wound healing which varies with the structure of the tissue. Recent research has investigated adjunctive therapy with cytotoxic drugs, chemical agents or irradiation as a means of enhancing the efficacy of cryosurgical treatment. Though experiments in vitro and clinical results are mentioned occasionally to support or verify the work in animals, this review focuses on the diverse aspects of cryosurgical research in vivo.
Section snippets
Direct cell injury
The cornerstone of direct cell injury from freezing is ice crystal formation which removes water from the cells and leads to a sequence of deleterious events [126], [132]. Recent investigations in vitro have identified apoptosis, or gene regulated cell death, as a mechanism of direct cell injury [89]. This was confirmed by experiments in vivo by Steinbach et al., showing that cell death is by necrosis in the central part of the cryogenic lesion and that apoptosis is evident in the peripheral
The vascular injury
Early in modern cryosurgery, many experiments with diverse tissues, such as rat liver, guinea pig skin, rabbit skin, and hamster cheek pouch, showed the sequence of events that follow freezing and thawing and ending in circulatory failure. When the tissue thaws, the previously frozen volume of tissue becomes congested and edematous, which extends progressively during the next few hours. The cause of the edema is endothelial cell damage, manifest as defects in endothelial cell junctions as early
Technology-methods and control of freezing
A large variety of cryogenic agents and freezing techniques with diverse apparatus have been used in cryosurgical experiments and treatment. Investigators in the first half of the 1900’s produced lesions by freezing various tissues and developed a considerable amount of knowledge pertinent to cryosurgery. These experiments, which defined the cryosurgical lesion, helped pave the way for the modern era of cryosurgery, initiated by Cooper and Lee in 1961 when they developed automated apparatus
The freeze–thaw cycle
An understanding of the mechanisms of cell and tissue injury requires recognition of the effects of the freeze–thaw cycle as used in cryosurgery. Every facet of the freeze–thaw cycle may produce injury to the tissue, and all may be manipulated. Therefore knowledge of the effect of each phase of the cycle is critical, whether the goal is complete or selective tissue destruction. Though the components of the freeze–thaw cycle are difficult to describe with precision in a frozen volume of tissue,
The cryogenic lesion
Before the modern era began in the 1960’s, local freezing techniques were used to destroy tissue, as in the brain, heart, liver, and kidney of rabbits and cats, for physiological studies [58]. These experiments accurately described the nature of the cryogenic lesion which investigators characterized as a volume of circumscribed necrosis, featuring a large central necrotic region surrounded by a narrow peripheral or border zone of partially damaged and surviving cells. The peripheral or border
Diverse tissue
The freezing of most tissues produces the typical cryogenic lesion of demarcated coagulation necrosis. The process of healing, though similar in cellular infiltrations and establishment of new blood supply, differs in the diverse tissues. The skin is high in collagen, elastin, and fibroblasts, which resist freezing injury, so healing is ordinarily favorable. Damaged collagen is absorbed and slowly replaced by new collagen. Muscle fibers and cellular tissue, such as the liver and kidney, heal
Adjunctive therapy
Many recent experiments are directed at improving the efficacy of cryosurgery. The greatest opportunity for improved efficacy is in the management of the periphery of the cryogenic lesion where cell destruction is partial. Therapy adjunctive to cryosurgery, such as chemical agents, cytotoxic drugs, or irradiation should prove helpful in completing destruction of cells in the periphery of the frozen volume.
Cancer chemotherapeutic drugs are the most commonly used adjunctive therapeutic agents.
Summary
Cryosurgery had its beginning in the mid 1850’s when irrigation with freezing solutions were used to treat advanced incurable cancer. Progress thereafter was slow in the first half of the 1900’s when liquid air and carbon dioxide were used for freezing tissue in the first half of the 1900’s. Modern cryosurgery began with the development of automated apparatus cooled by liquid nitrogen early in the 1960’s. At that time, the major source of knowledge pertinent to cryosurgery was the prior and
Acknowledgments
The authors would also like to recognize Christine Baust’s efforts in the preparation and revision of this manuscript.
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Statement of funding: Preparation of this manuscript was supported in part by NIH — National Cancer Institute Grant #’s 1R43CA123993-01A1, 1R43CA128357-01A2, and 1R43CA118537-02.