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Fundamental Aspects of Cryosurgery

John Baust1 MO Maiwand 2.
1Institute of Biomedical Technology
SUNY Binghamton, Binghamton, NY 13902 USA.
2Harefield Hospital, Harefield, UK.

During the 1990's three technological developments led to a rebirth of interest in cryosurgery. These included the improvement in cryosurgical

instrumentation, the use of intra-operative ultrasound to view the formation of ice crystals within the tissue in real time and the development of minimally invasive surgical techniques. More importantly, there has been a renewed interest in defining the mechanisms of cell death following cryosurgery.

The Mechanism of Injury: It has been widely recognized for many years that two major mechanisms of injury contribute to cryosurgical-effect of cell death. These mechanisms include direct injury to cells due to ice crystal formation, especially intracellular damage, and microcirculatory failure due to capillary destruction. A comprehensive review of these mechanisms has been provided by Gage and Baust (1998). More recent advances have linked the fundamentals of cryosurgery and cryopreservation of tissues and organs to stimulate new interest in direct cell injury.

Cell, tissue and organ preservation and cryosurgery share the same problem with ice crystal formation. The cell membrane functions as a barrier to extracellular ice penetration, so the manner in which ice enters the cell is of interest and the topic of recent research (Karlson, et al. 1993). The membrane - ice interface and the dynamics of these two components have proven to be a factor critical to cell survival. The freeze concentration of solutes in the liquid barrier between the crystalline ice (pure water) and the hydrophobic cell membrane results in a hyperosmotic solution thought to stress the cell in a manner that might either injure the cell membrane sufficiently to permit the entry of ice (Muldrew and McGann, 1994) or provoke cell death through a signal transduction process. The latter would result in the initiation of the apoptotic cell death cascade (Clarke, et al. 2001). Additional issues of cryosurgical-dependent injury relate to the role of ice propagation through cell-to-cell contacts and the physical disruption to tissue structure caused by ice propagation.

Arguably, the most important advance in basic research related to cryosurgery is the recognition that apoptosis, gene regulated cell death, is a mechanism of injury following tissue freezing. While the necrotic boundary is sharply circumscribed in a cryogenic lesion, the periphery of the lesion, the border zone, does not experience sufficiently low temperatures to be lethal to all cells. In this margin of the cryolesion, some cells survive, linger for a few days and then die through a process defined as apoptosis (Hollister, et al. 1998). Cells in this region show evidence of non-random DNA cleavage, membrane blebbing, membrane phospholipid inversion and caspase activation (Clarke et al., 1999, 2000). Cells exposed to lower temperatures consistent with the central lesion mass demonstrate cell death characteristics common to necrosis.

The observation of a complex molecular biological event (apoptosis) occurring in the region of likely cryosurgical failure, and therefore possible disease recurrence, has clinical implications since most chemotherapeutic agents also work through this same destructive pathway. In recent studies on human prostate cancer cells (PC-3) it was noted that cells exposed to -25°to -80°C yielded 1- 10% survival and only when cells were exposed to -100°C was complete mortality realized. However, if PC-3 cells were first exposed to a sub-clinical dose of 5-fluorouracil or other cytotoxic agents, complete loss of viability was obtained between -5° and -25°C. These result show both are remarkable resistance of these cancer cells to freezing and suggest that combination chemo-cryotherapy is of potential therapeutic benefit. Similar observations have been made using a human colon carcinoma cell line (Hanai et al., 2001).


 

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