- Email: [email protected]
A study of infrared thermographic assessment of liquid nitrogen cryotherapy
A study of infrared thermographic assessment of liquid nitrogen cryotherapy M. Anthony Pogrel, DDS, MD, a Chung Kwan Yen, DDS, b and Robert Taylor, DDS, MDS, c San Francisco, Calif. DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY, UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Objective. To assess whether infrared thermography can accurately predict the area of soft tissue necrosis that results from liquid nitrogen cryoprobe therapy. Study design. Eight rats received cryosurgery on the shaved abdomen with a liquid nitrogen cryoprobe in a triple-freeze technique. The therapy was monitored with infrared thermography and thermocouple probes. The temperature reached was correlated with the area of tissue necrosis found on histologic examination at sacrifice 1 week after the cryotreatment. A parallel study was carried out on pieces of beefsteak to assess the depth and shape of freeze. Results. The -20~ isotherm, which is felt to correspond to the cell lethal zone, occupied the inner 70% of the area of the iceball produced. Histologically, the -20~ isotherm corresponded well to the area of tissue necrosis. In depth, the iceball takes on a semicircular shape. Conclusions. Infrared tomography is expensive to use clinically and cannot be readily used in the oral cavity. However, this study does show that one can clinically estimate that the inner 70% of the area of an iceball produced by liquid nitrogen on soft tissues will ultimately undergo necrosis. (ORAL SURGORAL MED ORAl. PA-fHOL ORAL RADIOI- ENDOD 1996;81;396-401)
It is known that intense cold can cause cell death. It was not, however, until 1961 that a controllable system was developed for use in surgical ablation of tissue.1 The use of open and closed systems has allowed the use of nitrous oxide, carbon dioxide, or liquid nitrogen as a freezing agent to cause tissue death. Liquid nitrogen, which boils at -196~ achieves a lower temperature than the other two modalities (nitrous oxide boils a t - 8 9 . 7 ~ and carbon dioxide at -78.5~ and therefore it can achieve a more profound freezing effect. There is, however, debate as to the temperature required to kill mammalian cells by means of cryotherapy and also the exact mode of cell death. Cell death appears to occur by a combination of direct damage from intracellular and extracellular ice crystal formation plus osmotic and electrolyte disturbances. 2 Temperatures below - 2 0 ~ are generally felt to cause consistent mammalian cell death) The edge of a visible iceball created during cryotherapy is at approximately -2~ and most authorities advocate multiple
This study was funded in part by NIH-BSRG grant RR-5305. aprofessor and Chairman. bAssistant Clinical Professor. cPt'ofessor Emeritus. Received for publication July, 17, 1995; returned for revision Oct. 6, 1995; accepted for publication Nov. 30, 1995. Copyright 9 1996 by Mosby-Year Book, Inc. 1079-2104196/$5.00 + 0 7/12/70998
396
overlapping freeze-thaw cycles at cell lethal temperatures to achieve maximum cell death. 4 This is because without accurate temperature monitoring within the tissues, there is uncertainty about the exact extent of the cell lethal zone. The general advice is to freeze rapidly to cause the maximum intracellular ice crystal formation and then allow the tissues to thaw slowly to cause the maximum electrolyte disturbance because both of these situations result in maximal cell lethal effects. 5 The temperatures reached in the tissues are affected by a number of variables; this makes accurate temperature calculation difficult. Nearby blood vessels can lessen the freezing effect by the so-called "heat-sink" phenomenon. Ice itself is a good thermal insulator and so prevents extensive spread of the thermal effects of cryosurgery. Current methods of temperature measurement include the arbitrary one of measuring the iceball and estimating that the inner two thirds will be below the cell lethal temperature and will be devitalized and that if overlapping iceballs are used, a wider cell death will occur. Thermocouples can be implanted into the tissues but suffer the disadvantage that they are position sensitive; also they can only record the temperature at one particular point. In addition, there is a theoretical risk of thermocouple probes displacing cells from a lesion to be treated by cryosurgery. Magnetic resonance imaging may represent a theoretical means of monitoring the extent of the frozen
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 81, Number 4
Pogrel, Yen, and Taylor
397
Fig. 1. Isotherm pattern of 1 cm round cryoprobe applied to shaved rat abdomen for 1 minute. The -20~ isotherm (the first blue isotherm) occupies 53% of area of the visible iceball (-2~ red isotherm).
area but it is unlikely to beome widely available for this purpose. 6 In the present study an infrared thermal imaging system was evaluated as a means of assessing the temperatures achieved with cryosurgery to correlate the temperatures reached with the clinical area of necrosis and also to assess the practicality of infrared thermography in clinical practice.
MATERIAL AND METHODS The Hughes Probeye 4300 Thermal Imaging System (Hughes Aircraft Co, Carlsbad, Calif.) was used for this study. This remote infrared thermographic camera can be accurately focused from a wide range of distances and the information stored on videotape. The equipment has a discrimination of O.I~ at ambient temperatures and 0.5~ down to -40~ which r e p r e s e n t s the lower limit of the equipment. The rat animal model was used for this study, and the National Research Council guide for the care of animals was adhered to. Eight 250 gm SpragueDawley rats were anesthetized with 25 mg/kg of intraperitoneal nembutal, and the abdomen was shaved. A Brymill (Brymill Corp., Vernon, Conn,) cryogenic liquid nitrogen cryosurgery apparatus was used to apply a 1 cm circular flat cryoprobe with an electrode gel coupling medium to the rat abdomen for 1 minute after the appearance of the iceball; the temperature was recorded thermographically. After 1 minute the cryoprobe was removed from the tissues that were then allowed to rewarm until a steady temperature
was reached. Two repeat freezes were applied to the same area with a similar period of rewarming between. All freezes were monitored with the thermal imaging system and recorded on videotape. Two conventional Fridgitronics (Fridgitronics Inc., Shelton, Conn.) thermocouples were also used (one on either side of the iceball) to compare the temperatures with those recorded by the infrared imaging system. The clinical margins of the largest iceball (-2~ were identified by tattooing with India ink. The rats were sacrificed 7 days after the cryotreatment at which time the area of tissue necrosis was cliiaically delineated. Blocks were taken from the cryolesion to include its boundaries, fixed in formalin, embedded, and sectioned. Six micron sections were cut and stained with hematoxylin and eosin. In a parallel study, eight blocks of beefsteak 3 cm in thickness were subjected to the identical cryotreatment as in the preceding study, but this was carried out on the edge of the steak so that the depth of the cryolesion could be visualized. The freezing in each case was monitored with the infrared thermal imaging system.
RESULTS After an initial 1 minute freeze with the cryoprobe on the rat abdomen, the mean diameter of the clinical iceball was 1.75 cm (range, 1.6 to 2.0 cm; n = 8). The thermographic a p p e a r a n c e of this initial iceball is shown in Fig. 1. In this figure, the -24~ isotherm is
398
Pogrel, Yen, and Taylor
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
April 1996
Fig. 2. Isotherm pattern 40 seconds after removal of probe. Coldest area is now at approximately -16~
the coldest recorded so that all blue areas are below -20~ which represents the cell lethal zone. The edge of the clinical iceball corresponds to the area between the -4~ and 0~ isotherms. The mean diameter of the 20~ isotherm is 1.5 cm (range, 1.1 cm to 1.7 cm; n = 8); the mean diameter of the -2~ isotherm is 1.8 cm (range, 1.6 to 2.4 cm). Thus if -20~ represents the cell lethal zone, in this study the cell lethal zone is 83% of the diameter of the clinical iceball and 69% of the area. Some of the isotherms are irregular because of the heat-sink effect of the surrounding soft tissues. The irregularities in the isotherm shape increase as the temperatures rise. After removal of the probe after the first freeze, it took a mean of 2.5 minutes for the temperature to rise above 0~ on the rat abdomen (range, 1.75 to 3 minutes; n = 8). The temperatures 40 seconds after removal of the probe are in Fig. 2 and show that parts of the treated area are still below -10~ With the repeat freeze-thaw cycle, the volume of tissue frozen is greater with each freeze. The diameter of the -20~ isotherm increased from a mean of 1.5 cm to a mean of 1.7 cm with the third freeze. On removal of the probe, the temperatures also take longer to rise above 0~ with successive freezes. On the third freeze, it took a mean of 3.7 minutes (range, 2.5 to 4.5 minutes; n = 8) for the temperature to rise above 0~ These results are summarized in Fig. 3. Comparison of the temperatures recorded by the infrared thermal imaging system and the thermocouple showed good temperature correlation. The infrared system has a sensitivity of 0.5~ whereas the
thermocouple has a sensitivity of 2~ Within these limits the difference between the two temperatures indicated was never more than plus or minus 3.0~ The histologic sections of the eight lesions on the rats show the final area of necrosis on the epithelial surface to be a mean of 1.5 cm in diameter (range, 1.3 to 1.7 cm, n = 8) and therefore to correspond well with the 20~ isotherm as a predictor of the final area of tissue necrosis on soft tissues. Criteria for tissue necrosis include nuclear pyknosis, nuclear fragmentation, karyolysis, nuclear karyolysis, and hyperchromatic cytoplasm. 7-9 Measurements were taken directly from the histologic slides and confirmed at xl0 magnification with a calibrated graticule eyepiece (Fig. 4) with appropriate correction made for shrinkage artifact in the histologic processing (5% in our laboratory). When the 1 cm flat cryoprobe was used for 1 minute on the edge of a piece of steak, a thermogram pattern as shown in Fig. 5 is produced, and the temperatures are shown down to the -40~ isotherm. This shows that for a flat probe the cell lethal zone (-20~ is roughly semicircular in shape with its base on the surface. The maximum depth of the -20~ isotherm is 0.5 cm (range, 0.4 to 0.7 cm, n = 8) or about 50% of the width of the iceball.
DISCUSSION Cryosurgery has been used on the skin and in the oral cavity for a variety of disorders but has never gained widespread acceptance. Recent articles on its use for soft tissue lesions in the oral cavity are
Pogrel, Yen, and Taylor 399
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 81, Number 4
DIAMETER IN CMS
1 N U M B E R OF F R E E Z E ..-m. VISIBLE ICEBALL
--e- -2 C 180THERM
~
-20 C ISOTHERM
Fig. 3. Size of treated area increases with each freeze; greater increase is between the first and second freezes. There is a proportionately greater increase in size of the visible iceball and -2~ isotherm than the 20~ isotherm. This phenomenon is probably due to thrombosis in blood vessels after the first freeze, lessening the heat-sink effect of blood vessels. The -2~ isotherm and the visible iceball are approximately congruent.
Fig. 4. Histologic section of area treated by 1 cm cryoprobe in a triple-freeze technique at sacrifice on the seventh day. The diameter of the necrotic area is 1.5 cm. Note the cellular disruption by ice crystals with little evidence of regeneration. (Hematoxylin-eosin stain; original magnification xl0.)
f e w . 10"14 A s the oral m u c o s a is moist, it presents an ideal surface for the c r y o p r o b e b e c a u s e a c o u p l i n g m e d i u m is not required. H o w e v e r , its use does require k n o w l e d g e o f the p h y s i c a l extent o f the freezing and necrotic process. B r a d l e y 15 s h o w e d infrared t h e r m o -
graphic studies o f c r y o s u r g e r y with concentration p l a c e d on the - 1 5 ~ and - 2 . 0 ~ isotherms. In the present study, concentration has b e e n p l a c e d on the isotherms b e l o w . 2 0 ~ b e c a u s e these temperatures are n e c e s s a r y to ensure cell death. Nevertheless, the
400
Pogrel, Yen, and Taylor
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
April 1996
Fig. 5. Isotherm down to -40~ around I cm flat cryoprobe applied to edge of steak for I minute and viewed from side. Horizontal cross hair corresponds to surface of meat. Note shape of blue area beneath surface with approximate semicircular shape and depth of one third of its width.
shapes o f the isotherms p r o d u c e d with the liquid nit r o g e n c r y o p r o b e are in g e n e r a l a g r e e m e n t w i t h those o b t a i n e d b y Bradley. 15 T h e - 2 0 ~ cell lethal i s o t h e r m occupies approxim a t e l y 83% o f the width o f the clinical i c e b a l l (2.0~ isotherm); this c o r r e s p o n d s well with the subjective a s s e s s m e n t that the inner t w o thirds o f a visible iceball will necrose. S e c o n d and third freezes p r o b a b l y achieve a s o m e w h a t larger freeze b e c a u s e o f h e m o stasis f r o m p r e c e d i n g freezes that negate the h e a t - s i n k effect. O n e d i s a d v a n t a g e o f infrared t h e r m o g r a p h y is that it can o n l y m o n i t o r surface t e m p e r a t u r e s and cannot therefore m e a s u r e the depth o f the freezing effect. H o w e v e r , the studies on b l o c k s o f steak do give an a p p r o x i m a t e i d e a o f the p r o b a b l e shape and size o f the subepithelial freeze once the size o f the surface freeze is known. In the living animal, one m a y a s s u m e that the size and depth o f the freeze w o u l d b e less than on the steak b e c a u s e o f the h e a t - s i n k effect o f n e a r b y b l o o d vessels and a l l o w a n c e s w o u l d n e e d to b e m a d e for this. Infrared t h e r m o g r a p h y w o u l d a p p e a r to b e an accurate m e a n s o f m o n i t o r i n g the t e m p e r a t u r e s r e a c h e d b y liquid nitrogen c r y o t h e r a p y on a flat surface and c o r r e s p o n d s w e l l with t e m p e r a t u r e s r e c o r d e d b y t h e r m o c o u p l e . H o w e v e r , the e q u i p m e n t is e x p e n s i v e and not g e n e r a l l y available, and it is technique sensitive and requires careful maintenance. E x c e p t in e x c e p t i o n a l circumstances, it cannot b e c o n s i d e r e d a clinical tool. In addition, its use within the oral cavity is restricted b y both difficulty o f access for the c a m -
era and also the fact that m o s t surfaces are not flat and w o u l d not b e at right angles to the camera. Also, o f course, the c a m e r a can o n l y visualize the surface o f a lesion, and any i n f o r m a t i o n r e g a r d i n g the depth o f freeze can o n l y be inferred. Nevertheless, this study does indicate that current clinical a d v i c e that the inner t w o thirds o f a l i q u i d nitrogen iceball will necrose are certainly safe and p r o b a b l y even conservative criteria, because, in this study, up to 83% o f the dia m e t e r o f the iceball and 69% o f the area o f the iceball s h o w e d final necrosis. This study therefore indicates the area o f necrosis that m a y b e e x p e c t e d in clinical practice w i t h the use o f liquid nitrogen cryotherapy. We thank the Hughes Aircraft Company and Mr. Ken Huselman, Product Line Manager, Imaging and Design Section, for the loan of the thermographic camera. REFERENCES
1. Sguazzi A, Bracco D. A historical account of the technical means used in cryotherapy. Minn Med 1974;65:3718-22. 2. Whittaker DK. Mechanisms of tissue destruction following cryosurgery. Ann R Coil Surg Eng 1984;66:313-8. 3. Smith JJ, Fraser F. An estimation of tissue damage and thermal history in the cryolesion. Cryobiology 1974; 11:139-47. 4. Gill W, Fraser J, Carter DC. Repeated freeze-thaw cycles in cryosurgery. Nature 1968;219:410-3. 5. LePivert PJ. Basic consideration of the cryolesion. In: Ablin RJ, ed. Handbook of cryosurgery. New York: Marcel Dekker, 1980:22. 6. Hong JS, Wong S, Pease G, Rubinsky B. MR assisted temperature calculations during cryyosurgery. Magn Reson Imaging 1994;12:1021-31. 7. Malinin TI, Perry VP. A review of tissue and organ viability assay. Cryobiology 1967;4:106-15. 8. Earle WR, Schilling EL. Production of malignancy in vitro:
Pogrel, Yen, and Taylor
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
401
Volume 81, Number 4
9. 10. 11. 12. 13.
continued description of cells at the glass interface of the cultures. J N a t Cancer Inst 1950;10:1067-03. Wright GP. An introduction to pathology, 3rd ed. London: Longwans, 1958;172. Emmings FG, Koepf SW, Gage AA. Cryotherapy for benign lesions of the oral cavity. J Oral Surg 1967;25:320-6. Leopard PJ. Cryosurgery and its application to oral surgery. Br J Oral Surg 1975;13:128-52. Gage AA. Current progress in cryosurgery. Cryobiology 1988;25:483-6. Rubinsky B, Onik G. Cryosurgery: advances in the application of low temperatures to medicine. J Refridg 1991; 14: t 90-9.
14. Poswillo DE. Applications of cryosurgery in dentistry. Dent Update 1978;5:27-38. 15. Bradley PF. Thermography as an aid to cryosurgery. Acta Thermographica 1977;2:83-90.
Reprint requests: M. Anthony Pogrel, DDS, MD Department of Oral and Maxillofacial Surgery University of California, San Francisco 521 Parnassus Avenue, C-522 San Francisco, CA 94143-0440
Objective. To assess whether infrared thermography can accurately predict the area of soft tissue necrosis that results from liquid nitrogen cryoprobe therapy. Study design. Eight rats received cryosurgery on the shaved abdomen with a liquid nitrogen cryoprobe in a triple-freeze technique. The therapy was monitored with infrared thermography and thermocouple probes. The temperature reached was correlated with the area of tissue necrosis found on histologic examination at sacrifice 1 week after the cryotreatment. A parallel study was carried out on pieces of beefsteak to assess the depth and shape of freeze. Results. The -20~ isotherm, which is felt to correspond to the cell lethal zone, occupied the inner 70% of the area of the iceball produced. Histologically, the -20~ isotherm corresponded well to the area of tissue necrosis. In depth, the iceball takes on a semicircular shape. Conclusions. Infrared tomography is expensive to use clinically and cannot be readily used in the oral cavity. However, this study does show that one can clinically estimate that the inner 70% of the area of an iceball produced by liquid nitrogen on soft tissues will ultimately undergo necrosis. (ORAL SURGORAL MED ORAl. PA-fHOL ORAL RADIOI- ENDOD 1996;81;396-401)
It is known that intense cold can cause cell death. It was not, however, until 1961 that a controllable system was developed for use in surgical ablation of tissue.1 The use of open and closed systems has allowed the use of nitrous oxide, carbon dioxide, or liquid nitrogen as a freezing agent to cause tissue death. Liquid nitrogen, which boils at -196~ achieves a lower temperature than the other two modalities (nitrous oxide boils a t - 8 9 . 7 ~ and carbon dioxide at -78.5~ and therefore it can achieve a more profound freezing effect. There is, however, debate as to the temperature required to kill mammalian cells by means of cryotherapy and also the exact mode of cell death. Cell death appears to occur by a combination of direct damage from intracellular and extracellular ice crystal formation plus osmotic and electrolyte disturbances. 2 Temperatures below - 2 0 ~ are generally felt to cause consistent mammalian cell death) The edge of a visible iceball created during cryotherapy is at approximately -2~ and most authorities advocate multiple
This study was funded in part by NIH-BSRG grant RR-5305. aprofessor and Chairman. bAssistant Clinical Professor. cPt'ofessor Emeritus. Received for publication July, 17, 1995; returned for revision Oct. 6, 1995; accepted for publication Nov. 30, 1995. Copyright 9 1996 by Mosby-Year Book, Inc. 1079-2104196/$5.00 + 0 7/12/70998
396
overlapping freeze-thaw cycles at cell lethal temperatures to achieve maximum cell death. 4 This is because without accurate temperature monitoring within the tissues, there is uncertainty about the exact extent of the cell lethal zone. The general advice is to freeze rapidly to cause the maximum intracellular ice crystal formation and then allow the tissues to thaw slowly to cause the maximum electrolyte disturbance because both of these situations result in maximal cell lethal effects. 5 The temperatures reached in the tissues are affected by a number of variables; this makes accurate temperature calculation difficult. Nearby blood vessels can lessen the freezing effect by the so-called "heat-sink" phenomenon. Ice itself is a good thermal insulator and so prevents extensive spread of the thermal effects of cryosurgery. Current methods of temperature measurement include the arbitrary one of measuring the iceball and estimating that the inner two thirds will be below the cell lethal temperature and will be devitalized and that if overlapping iceballs are used, a wider cell death will occur. Thermocouples can be implanted into the tissues but suffer the disadvantage that they are position sensitive; also they can only record the temperature at one particular point. In addition, there is a theoretical risk of thermocouple probes displacing cells from a lesion to be treated by cryosurgery. Magnetic resonance imaging may represent a theoretical means of monitoring the extent of the frozen
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 81, Number 4
Pogrel, Yen, and Taylor
397
Fig. 1. Isotherm pattern of 1 cm round cryoprobe applied to shaved rat abdomen for 1 minute. The -20~ isotherm (the first blue isotherm) occupies 53% of area of the visible iceball (-2~ red isotherm).
area but it is unlikely to beome widely available for this purpose. 6 In the present study an infrared thermal imaging system was evaluated as a means of assessing the temperatures achieved with cryosurgery to correlate the temperatures reached with the clinical area of necrosis and also to assess the practicality of infrared thermography in clinical practice.
MATERIAL AND METHODS The Hughes Probeye 4300 Thermal Imaging System (Hughes Aircraft Co, Carlsbad, Calif.) was used for this study. This remote infrared thermographic camera can be accurately focused from a wide range of distances and the information stored on videotape. The equipment has a discrimination of O.I~ at ambient temperatures and 0.5~ down to -40~ which r e p r e s e n t s the lower limit of the equipment. The rat animal model was used for this study, and the National Research Council guide for the care of animals was adhered to. Eight 250 gm SpragueDawley rats were anesthetized with 25 mg/kg of intraperitoneal nembutal, and the abdomen was shaved. A Brymill (Brymill Corp., Vernon, Conn,) cryogenic liquid nitrogen cryosurgery apparatus was used to apply a 1 cm circular flat cryoprobe with an electrode gel coupling medium to the rat abdomen for 1 minute after the appearance of the iceball; the temperature was recorded thermographically. After 1 minute the cryoprobe was removed from the tissues that were then allowed to rewarm until a steady temperature
was reached. Two repeat freezes were applied to the same area with a similar period of rewarming between. All freezes were monitored with the thermal imaging system and recorded on videotape. Two conventional Fridgitronics (Fridgitronics Inc., Shelton, Conn.) thermocouples were also used (one on either side of the iceball) to compare the temperatures with those recorded by the infrared imaging system. The clinical margins of the largest iceball (-2~ were identified by tattooing with India ink. The rats were sacrificed 7 days after the cryotreatment at which time the area of tissue necrosis was cliiaically delineated. Blocks were taken from the cryolesion to include its boundaries, fixed in formalin, embedded, and sectioned. Six micron sections were cut and stained with hematoxylin and eosin. In a parallel study, eight blocks of beefsteak 3 cm in thickness were subjected to the identical cryotreatment as in the preceding study, but this was carried out on the edge of the steak so that the depth of the cryolesion could be visualized. The freezing in each case was monitored with the infrared thermal imaging system.
RESULTS After an initial 1 minute freeze with the cryoprobe on the rat abdomen, the mean diameter of the clinical iceball was 1.75 cm (range, 1.6 to 2.0 cm; n = 8). The thermographic a p p e a r a n c e of this initial iceball is shown in Fig. 1. In this figure, the -24~ isotherm is
398
Pogrel, Yen, and Taylor
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
April 1996
Fig. 2. Isotherm pattern 40 seconds after removal of probe. Coldest area is now at approximately -16~
the coldest recorded so that all blue areas are below -20~ which represents the cell lethal zone. The edge of the clinical iceball corresponds to the area between the -4~ and 0~ isotherms. The mean diameter of the 20~ isotherm is 1.5 cm (range, 1.1 cm to 1.7 cm; n = 8); the mean diameter of the -2~ isotherm is 1.8 cm (range, 1.6 to 2.4 cm). Thus if -20~ represents the cell lethal zone, in this study the cell lethal zone is 83% of the diameter of the clinical iceball and 69% of the area. Some of the isotherms are irregular because of the heat-sink effect of the surrounding soft tissues. The irregularities in the isotherm shape increase as the temperatures rise. After removal of the probe after the first freeze, it took a mean of 2.5 minutes for the temperature to rise above 0~ on the rat abdomen (range, 1.75 to 3 minutes; n = 8). The temperatures 40 seconds after removal of the probe are in Fig. 2 and show that parts of the treated area are still below -10~ With the repeat freeze-thaw cycle, the volume of tissue frozen is greater with each freeze. The diameter of the -20~ isotherm increased from a mean of 1.5 cm to a mean of 1.7 cm with the third freeze. On removal of the probe, the temperatures also take longer to rise above 0~ with successive freezes. On the third freeze, it took a mean of 3.7 minutes (range, 2.5 to 4.5 minutes; n = 8) for the temperature to rise above 0~ These results are summarized in Fig. 3. Comparison of the temperatures recorded by the infrared thermal imaging system and the thermocouple showed good temperature correlation. The infrared system has a sensitivity of 0.5~ whereas the
thermocouple has a sensitivity of 2~ Within these limits the difference between the two temperatures indicated was never more than plus or minus 3.0~ The histologic sections of the eight lesions on the rats show the final area of necrosis on the epithelial surface to be a mean of 1.5 cm in diameter (range, 1.3 to 1.7 cm, n = 8) and therefore to correspond well with the 20~ isotherm as a predictor of the final area of tissue necrosis on soft tissues. Criteria for tissue necrosis include nuclear pyknosis, nuclear fragmentation, karyolysis, nuclear karyolysis, and hyperchromatic cytoplasm. 7-9 Measurements were taken directly from the histologic slides and confirmed at xl0 magnification with a calibrated graticule eyepiece (Fig. 4) with appropriate correction made for shrinkage artifact in the histologic processing (5% in our laboratory). When the 1 cm flat cryoprobe was used for 1 minute on the edge of a piece of steak, a thermogram pattern as shown in Fig. 5 is produced, and the temperatures are shown down to the -40~ isotherm. This shows that for a flat probe the cell lethal zone (-20~ is roughly semicircular in shape with its base on the surface. The maximum depth of the -20~ isotherm is 0.5 cm (range, 0.4 to 0.7 cm, n = 8) or about 50% of the width of the iceball.
DISCUSSION Cryosurgery has been used on the skin and in the oral cavity for a variety of disorders but has never gained widespread acceptance. Recent articles on its use for soft tissue lesions in the oral cavity are
Pogrel, Yen, and Taylor 399
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 81, Number 4
DIAMETER IN CMS
1 N U M B E R OF F R E E Z E ..-m. VISIBLE ICEBALL
--e- -2 C 180THERM
~
-20 C ISOTHERM
Fig. 3. Size of treated area increases with each freeze; greater increase is between the first and second freezes. There is a proportionately greater increase in size of the visible iceball and -2~ isotherm than the 20~ isotherm. This phenomenon is probably due to thrombosis in blood vessels after the first freeze, lessening the heat-sink effect of blood vessels. The -2~ isotherm and the visible iceball are approximately congruent.
Fig. 4. Histologic section of area treated by 1 cm cryoprobe in a triple-freeze technique at sacrifice on the seventh day. The diameter of the necrotic area is 1.5 cm. Note the cellular disruption by ice crystals with little evidence of regeneration. (Hematoxylin-eosin stain; original magnification xl0.)
f e w . 10"14 A s the oral m u c o s a is moist, it presents an ideal surface for the c r y o p r o b e b e c a u s e a c o u p l i n g m e d i u m is not required. H o w e v e r , its use does require k n o w l e d g e o f the p h y s i c a l extent o f the freezing and necrotic process. B r a d l e y 15 s h o w e d infrared t h e r m o -
graphic studies o f c r y o s u r g e r y with concentration p l a c e d on the - 1 5 ~ and - 2 . 0 ~ isotherms. In the present study, concentration has b e e n p l a c e d on the isotherms b e l o w . 2 0 ~ b e c a u s e these temperatures are n e c e s s a r y to ensure cell death. Nevertheless, the
400
Pogrel, Yen, and Taylor
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
April 1996
Fig. 5. Isotherm down to -40~ around I cm flat cryoprobe applied to edge of steak for I minute and viewed from side. Horizontal cross hair corresponds to surface of meat. Note shape of blue area beneath surface with approximate semicircular shape and depth of one third of its width.
shapes o f the isotherms p r o d u c e d with the liquid nit r o g e n c r y o p r o b e are in g e n e r a l a g r e e m e n t w i t h those o b t a i n e d b y Bradley. 15 T h e - 2 0 ~ cell lethal i s o t h e r m occupies approxim a t e l y 83% o f the width o f the clinical i c e b a l l (2.0~ isotherm); this c o r r e s p o n d s well with the subjective a s s e s s m e n t that the inner t w o thirds o f a visible iceball will necrose. S e c o n d and third freezes p r o b a b l y achieve a s o m e w h a t larger freeze b e c a u s e o f h e m o stasis f r o m p r e c e d i n g freezes that negate the h e a t - s i n k effect. O n e d i s a d v a n t a g e o f infrared t h e r m o g r a p h y is that it can o n l y m o n i t o r surface t e m p e r a t u r e s and cannot therefore m e a s u r e the depth o f the freezing effect. H o w e v e r , the studies on b l o c k s o f steak do give an a p p r o x i m a t e i d e a o f the p r o b a b l e shape and size o f the subepithelial freeze once the size o f the surface freeze is known. In the living animal, one m a y a s s u m e that the size and depth o f the freeze w o u l d b e less than on the steak b e c a u s e o f the h e a t - s i n k effect o f n e a r b y b l o o d vessels and a l l o w a n c e s w o u l d n e e d to b e m a d e for this. Infrared t h e r m o g r a p h y w o u l d a p p e a r to b e an accurate m e a n s o f m o n i t o r i n g the t e m p e r a t u r e s r e a c h e d b y liquid nitrogen c r y o t h e r a p y on a flat surface and c o r r e s p o n d s w e l l with t e m p e r a t u r e s r e c o r d e d b y t h e r m o c o u p l e . H o w e v e r , the e q u i p m e n t is e x p e n s i v e and not g e n e r a l l y available, and it is technique sensitive and requires careful maintenance. E x c e p t in e x c e p t i o n a l circumstances, it cannot b e c o n s i d e r e d a clinical tool. In addition, its use within the oral cavity is restricted b y both difficulty o f access for the c a m -
era and also the fact that m o s t surfaces are not flat and w o u l d not b e at right angles to the camera. Also, o f course, the c a m e r a can o n l y visualize the surface o f a lesion, and any i n f o r m a t i o n r e g a r d i n g the depth o f freeze can o n l y be inferred. Nevertheless, this study does indicate that current clinical a d v i c e that the inner t w o thirds o f a l i q u i d nitrogen iceball will necrose are certainly safe and p r o b a b l y even conservative criteria, because, in this study, up to 83% o f the dia m e t e r o f the iceball and 69% o f the area o f the iceball s h o w e d final necrosis. This study therefore indicates the area o f necrosis that m a y b e e x p e c t e d in clinical practice w i t h the use o f liquid nitrogen cryotherapy. We thank the Hughes Aircraft Company and Mr. Ken Huselman, Product Line Manager, Imaging and Design Section, for the loan of the thermographic camera. REFERENCES
1. Sguazzi A, Bracco D. A historical account of the technical means used in cryotherapy. Minn Med 1974;65:3718-22. 2. Whittaker DK. Mechanisms of tissue destruction following cryosurgery. Ann R Coil Surg Eng 1984;66:313-8. 3. Smith JJ, Fraser F. An estimation of tissue damage and thermal history in the cryolesion. Cryobiology 1974; 11:139-47. 4. Gill W, Fraser J, Carter DC. Repeated freeze-thaw cycles in cryosurgery. Nature 1968;219:410-3. 5. LePivert PJ. Basic consideration of the cryolesion. In: Ablin RJ, ed. Handbook of cryosurgery. New York: Marcel Dekker, 1980:22. 6. Hong JS, Wong S, Pease G, Rubinsky B. MR assisted temperature calculations during cryyosurgery. Magn Reson Imaging 1994;12:1021-31. 7. Malinin TI, Perry VP. A review of tissue and organ viability assay. Cryobiology 1967;4:106-15. 8. Earle WR, Schilling EL. Production of malignancy in vitro:
Pogrel, Yen, and Taylor
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
401
Volume 81, Number 4
9. 10. 11. 12. 13.
continued description of cells at the glass interface of the cultures. J N a t Cancer Inst 1950;10:1067-03. Wright GP. An introduction to pathology, 3rd ed. London: Longwans, 1958;172. Emmings FG, Koepf SW, Gage AA. Cryotherapy for benign lesions of the oral cavity. J Oral Surg 1967;25:320-6. Leopard PJ. Cryosurgery and its application to oral surgery. Br J Oral Surg 1975;13:128-52. Gage AA. Current progress in cryosurgery. Cryobiology 1988;25:483-6. Rubinsky B, Onik G. Cryosurgery: advances in the application of low temperatures to medicine. J Refridg 1991; 14: t 90-9.
14. Poswillo DE. Applications of cryosurgery in dentistry. Dent Update 1978;5:27-38. 15. Bradley PF. Thermography as an aid to cryosurgery. Acta Thermographica 1977;2:83-90.
Reprint requests: M. Anthony Pogrel, DDS, MD Department of Oral and Maxillofacial Surgery University of California, San Francisco 521 Parnassus Avenue, C-522 San Francisco, CA 94143-0440