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
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.,