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10.1245/ASO.2005.04.014
Annals of Surgical Oncology 12:303-312 (2005)
© 2005 Society of Surgical Oncology
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Original Article

Arteriolovenular Shunting Critically Determines Shutdown of Microcirculation Upon Cryotherapy in Tumor-Bearing Rat Liver

Sven Richter, MD1, Georg Pistorius, MD1, Roland Seidel, MD2, Gernot Feifel, MD1, Martin K. Schilling, MD1, Brigitte Vollmar, MD3,4 and Michael D. Menger, MD3

1 Department for General, Visceral, and Vascular Surgery, University of Saarland, D-66421 Homburg/Saar, Germany
2 Department of Radiology, University of Saarland, D-66421, Homburg/Saar, Germany
3 Institute for Clinical & Experimental Surgery, University of Saarland, D-66421, Homburg/Saar, Germany
4 Department of Experimental Surgery, University of Rostock, D-18055, Rostock, Germany

Correspondence: Address correspondence and reprint requests to: Michael D. Menger, MD; E-mail: exmdme{at}uniklinik-saarland.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Tissue destruction by cryosurgery not only is mediated by direct cell damage, but also involves secondary mechanisms, such as ischemia due to shutdown of the microcirculation. Clinicians favor repetitive cryoapplication, although there is no proven evidence for a more effective tumor eradication.

Methods: The aims of this study were (1) to establish a rat liver tumor model that allows for intravital microscopic analysis of hepatic tumor microcirculation and (2) to elucidate critical determinants of shutdown of microvascular perfusion after single and repetitive cryotherapy. In WAG-Rji rats (n = 14), syngeneic colon carcinoma cells (CC531) were implanted into the left liver lobe. Hepatic and tumor microcirculation were studied by intravital microscopy.

Results: Two weeks after implantation, the tumors had developed a microvasculature with a capillary density markedly (P < .05) lower compared with the sinusoidal density of normal liver. However, at the tumor margin, venule diameters were significantly enlarged (P < .05), with high red blood cell velocities and arteriolovenular shunts. Both freeze procedures (temperature at the tumor margin: –32.4°C ± 1.6°C and –36.4°C ± 2.0°C) resulted in a complete shutdown of intratumoral and peritumoral capillary and hepatic sinusoidal perfusion. In contrast, some large venules showed maintenance of blood flow initially after freezing (15 minutes); however, this was abolished during the subsequent 2-hour observation period.

Conclusions: Enlarged high-flow venules at the tumor margin, which participate in arteriolovenular shunting, critically determine the shutdown of the microcirculation upon cryotherapy. Repetitive freezing is not more effective than a single-freeze procedure to achieve complete tumor microcirculatory stasis.

Key Words: Cryotherapy • Cryosurgery • Microcirculation • Hepatic carcinoma • Hepatic metastasis • Colon carcinoma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A considerable number of patients with colon carcinoma experience either synchronous or meta-chronous hepatic metastasis, but only 10% to 40% of these patients are suitable for surgical resection.1,2 For those with nonresectable liver tumors, the only therapeutic options are palliative chemotherapy or local tumor-destruction techniques. Because the mean life expectancy after systemic chemotherapy for hepatic metastases is limited to 9 to 17 months3,4 and because locoregional intra-arterial chemotherapy does not significantly improve these results,3,5,6 local tumor destruction by radiofrequency, laser-induced thermotherapy, or cryosurgery may be the only treatment option.

Although cryosurgery was introduced clinically in 19637 and was established for the treatment of liver malignomas in 1988,8 the underlying mechanisms of tissue destruction are still not fully understood. In consequence, optimal freeze modes and tumor-destruction conditions have not yet been determined. Intracellular formation of ice crystals has been assumed as the primary cause of cryotherapy-associated tumor cell death,9,10 but additional studies have indicated that the effectiveness of cryothermia may be enhanced by interrupting blood perfusion.11–13 In fact, cryothermia per se is thought to result in secondary injury by inducing microvascular perfusion failure and, thus, tissue anoxia.14–16

Malignant tissues have been suggested to exert a different response to freezing than normal tissues. Tumor cells of liver metastases have been shown to be more resistant against low temperatures than healthy liver tissue,17 probably because of a lower susceptibility to cell dehydration. In parallel, the malignant tissue microvasculature may also respond differently to freezing than the microvasculature of the healthy liver tissue. Recent in vivo studies have evidenced that in normal liver, a temperature of 0°C is sufficient to completely shut down the hepatic microcirculation, resulting in cell death and tissue necrosis.16,18 The freeze temperature required to shut down the tumor microvasculature, however, has not yet been determined.

To achieve sufficiently low temperatures at the margin of liver tumors, repetitive application of cryothermia was introduced. This is thought to result in a more homogeneous distribution of temperature decrease within the freezing zone. However, there is still no proven evidence for a more effective tumor eradication compared with single-freeze procedures. In addition, there are indications that repetitive freezing increases the rate of severe postoperative complications, probably because of more pronounced liver cell damage.19 Thus, the value of repetitive freezing remains to be determined. The aim of this study was therefore (1) to establish a rat liver tumor model that allows the intravital microscopic analysis of hepatic tumor microcirculation and (2) to elucidate the temperature and structural determinants adequate to shut down microvascular perfusion after single and repetitive cryotherapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
After approval of the local governmental animal care committee, experiments were performed in accordance with the German legislation on protection of animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication No. 86–23; revised 1985).

Tumor Model
Under ether anesthesia, 14 male WAG-Rji rats (weight, 260 ± 8 g; Charles River, Wiga, Sulzfeld, Germany) were laparotomized, and 1 x 106 tumor cells of a syngeneic colon carcinoma (CC531) were implanted under the capsule of the left liver lobe. At 14 days after tumor cell implantation, animals were anesthetized with pentobarbital (50 mg/kg body weight) and placed on an electronically regulated heating pad, which adjusted the body temperature to 37°C. After relaparotomy, the left liver lobe was mobilized by dissection of the ligaments and then placed on a three-dimensionally adjustable plasticine stage to allow application of cryotherapy and intravital fluorescence microscopy.16 In addition, polyethylene catheters (PE-50; .58-mm inner diameter; Portex, Hythe, UK) were inserted into the right carotid artery and the right jugular vein. This allowed for continuous monitoring of macro-hemodynamics (mean arterial pressure and heart rate), fluid substitution (saline 6 mL/kg/hour), and application of fluorochromes for intravital fluorescence microscopy.

Cryothermia
Perpendicular contact freezing was performed with a nitrogen-cooled cryoprobe (diameter, 3.2 mm; –180°C; Erbe, Tübingen, Germany), which was placed at the center of the tumor and which was connected to the cryosystem CRYO 6 (Erbe). The freezing temperature was measured by means of thermoprobes (diameter, 200 µm; Erbe) at the tip of the cryoprobe (center of the tumor), at the tumor margin, and at 3 mm outside of the tumor margin. Freezing was stopped when 0°C at 3 mm outside of the tumor margin was achieved, and this was followed by passive thawing (n = 7). Repetitive freezing (n = 7) included a second freeze cycle, which was started when passive thawing after the first cycle showed 0°C in the tumor center.

Intravital Fluorescence Microscopy
The animals on the heating pad were transferred to the stage of a modified Zeiss-Axiotech microscope (Zeiss, Oberkochen, Germany), which was equipped with a mercury arc lamp (100 W) and a blue filter block (excitation wavelength, 450–490 nm; emission wavelength, >515 nm; Zeiss) for epiillumination fluorescent light microscopy. Microscopic images were registered by a charge-coupled device video camera (Cohu FK 6990; Pieper, Schwerte, Germany) and were transferred to a video system. Magnifications of x 140 and x 350 were achieved on a 14-inch video screen. Sodium fluorescein (molecular weight, 376; 2 µmol/kg body weight) was applied intravenously to produce adequate negative contrast enhancement of the microvasculature for high-quality-resolution images.20 This allowed quantitative offline analysis of microcirculatory parameters by using a computer-assisted image-analysis system (CapImage; Zeintl, Heidelberg, Germany).

Microcirculatory Parameters
Because of differences in angioarchitecture, microcirculatory parameters within the areas of the tumor center and the tumor margin were analyzed separately and were compared with the normal adjacent hepatic microcirculation. In each animal, at least eight regions of interest were studied in the tumor center, the tumor margin, and the adjacent hepatic tissue. Microcirculatory parameters included the functional density of hepatic sinusoids and tumor capillaries; the diameters, tortuosity, and density of venules; the heterogeneity of venular density; and the venular red blood cell velocity and volumetric blood flow. Functional sinusoidal and capillary density were analyzed by counting the number of perfused sinusoids/capillaries crossing a 200-µm raster line (given in n/200 µm).21 Venular diameters were measured perpendicularly to the vessel path and are given in micrometers. The venular tortuosity was calculated by the ratio of the actual path length and the straight distance between the two visible end points of the venule.22 The venular density was determined by counting the number of vessels crossing a defined raster line and is given in n/mm. The heterogeneity of venular density, reflected by the coefficient of variation, was calculated by the standard deviation divided by the mean.23 Venular red blood cell velocity was determined by the use of the line shift method, incorporated in the software package of CapImage.24 Venular blood flow (Q) was calculated from venular diameters (D) and red blood cell velocities (VRBC) according to Q = {pi} x (D/2)2 x VRBC.

Experimental Protocol
Seven animals were studied with single freezing of the liver tumor, and seven additional animals underwent a repetitive (double) freeze procedure. Microcirculatory analyses were performed before application of cryothermia and were repeated in identical regions of interest initially (15 minutes) as well as 60 and 120 minutes after cryotherapy. During the freeze procedure, the tissue temperature at the tumor margin was monitored, and the effective freezing time (0°C at 3 mm outside of the tumor margin) was recorded and compared between the single- and repetitive-freeze modes.

Statistical Analysis
All values are expressed as means ± SEM. After the assumption of normality and homogeneity of variance were proven, differences between groups were calculated with a one-way analysis of variance and an unpaired Student’s t-test, including correction of the {alpha}-error according to Bonferroni probabilities. Differences within groups over time were assessed by one-way analysis of variance for repeated measurements, followed by the Student-Newman-Keuls test, which compensates for multiple comparisons. Overall statistical significance was set at P < .05. Statistical analysis was performed with SigmaStat (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor Growth and Vascularization
Relaparotomy at 14 days after tumor implantation revealed, in all 14 animals, the development of round liver tumors with a mean surface area of 25.1 ± 4.7 mm2. None of the animals showed signs of peritoneal or other extrahepatic metastases. Macrohemodynamics were in the reference range and remained stable throughout the experiments, including the period of cryotherapy (data not shown).

Intravital microscopy of hepatic and tumor microcirculation revealed a chaotic arrangement of newly formed tumor capillaries which was completely different from the regular arrangement of the sinusoids in the hepatic tissue adjacent to the tumor (Fig. 1Go). The capillary density in the center of the tumor was slightly but not significantly lower when compared with that of the tumor margin, whereas the nutritive microvasculature of the tumor-adjacent liver tissue showed a significantly higher sinusoidal density (P < .05; Fig. 2Go).



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  		FIG. 1. Intravital fluorescence microscopy of the chaotically arranged, newly formed microvascular network of CC531 colon tumors at 2 weeks after tumor cell implantation under the capsule of the left liver lobe. (A–C) Lower magnification and (D–F) high-magnification imaging of the microvasculature of the normal liver (A and D), the tumor margin (B and E), and the tumor center (C and F). Contrast enhancement was achieved by sodium fluorescein 2 µmol/kg. Original magnification, x 10 (A–C) and x 250 (D–F).

 


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  		FIG. 2. Functional tumor capillary and hepatic sinusoidal density within the tumor center, the tumor margin, and the tumor-adjacent hepatic tissue at 2 weeks after tumor cell implantation under the capsule of the left liver lobe. Data are mean ± SEM; *P < .05 versus tumor-adjacent hepatic tissue (liver). tu, tumor.

 
The analysis of venular density did not show significant differences among the tumor center, tumor margin, and healthy liver tissue (Fig. 3AGo). However, the calculation of the coefficient of variation revealed a significantly (P < .05) higher heterogeneity of venular density in the tumor margin when compared with the tumor center and the tumor-adjacent liver tissue (Fig. 3BGo). In fact, intravital microscopy showed large, tortuous venules at the tumor margin that were draining the blood from the tumor tissue to the normal liver parenchyma (Fig. 4AGo). The tortuosity of these venules was markedly higher (P < .05), not only in the tumor margin, but also in the tumor center, compared with healthy liver tissue (Fig. 4BGo). Further analysis revealed significantly enlarged venular diameters (P < .05) within the tumor margin (72.0 ± 5.6 µm) when compared with those of the tumor center (42.4 ± 1.8 µm) and the normal liver tissue (41.0 ± 3.4 µm; Fig. 5AGo). Finally, red blood cell velocity was significantly (P < .05) higher in venules of the tumor margin (.39 ± .04 mm/s) than in venules of the tumor center (.29 ± .02 mm/s; Fig. 5BGo). This resulted in a markedly (P < .05) enhanced volumetric blood flow in these microvessels (Fig. 5CGo), in which arteriolovenular shunt perfusion was occasionally observed (Fig. 4AGo).



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  		FIG. 3. Venular density (A) and coefficient of variation of venular density (B) within the tumor center, the tumor margin, and the tumor-adjacent hepatic tissue at 2 weeks after tumor cell implantation under the capsule of the left liver lobe. Data are mean ± SEM; #P < .05 versus the tumor center. tu, tumor.

 


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  		FIG. 4. Intravital microscopy of the hepatic tumor microvasculature, demonstrating the pronounced tortuosity of the draining venules (A). Note the arrangement of an arteriolovenular connection (arrows), which allows arteriolovenular shunt perfusion. Original magnification, x 25. (B) Quantitative analysis of the venular tortuosity (length/distance) within the tumor center, the tumor margin, and the tumor-adjacent hepatic tissue at 2 weeks after tumor cell implantation under the capsule of the left liver lobe. Data are mean ± SEM; *P < .05 versus tumor-adjacent hepatic tissue (liver). tu, tumor.

 


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  		FIG. 5. Venular diameters (A), venular red blood cell velocity (B), and venular volumetric blood flow (C) within the tumor center, the tumor margin, and the tumor-adjacent hepatic tissue at 2 weeks after tumor cell implantation under the capsule of the left liver lobe. Data are mean ± SEM; *P < .05 versus tumor-adjacent hepatic tissue (liver); #P < .05 versus the tumor center. tu, tumor.

 
Cryotherapy and Tumor Microcirculation
According to the experimental protocol, the effect of the single-freeze procedure in liver tumors was compared with that of repetitive freezing (n = 7 each). Whereas the tumor size did not differ significantly between the twogroups (single freezing, 28.8 ± 8.2 mm2; repetitive freezing, 21.9 ± 5.5 mm2), the effective freezing time was almost doubled by the repetitive-freeze procedure (single freezing, 93.8 ± 11.4 s; repetitive freezing, 162.3 ± 13.2 s; P < .05). The final tissue temperature at the tumor margin was –32.4°C ± 1.6°C after single freezing and –32.2°C ± 1.2°C and –36.4°C ± 2.0°C after the first and the second cycles of the double-freeze procedure.

Single and repetitive freezing resulted in an immediate and complete shutdown of tumor capillary and hepatic sinusoidal perfusion, as indicated by a functional density of these microvessels of 0, including the tumor center, the tumor margin, and the 3-mm zone of the surrounding hepatic tissue (Fig. 6Go). The single-freeze procedure, but not repetitive freezing, further induced a significant vasodilation in the tumor margin (Fig. 7Go), as indicated by a 25% increase of venular diameters compared with baseline at 120 minutes after cryotherapy (P < .05). Analysis of venular perfusion at 15 minutes after cryotherapy revealed in all animals a complete shutdown in the tumor center and the tumor-adjacent hepatic tissue but maintained perfusion in some venules in the tumor margin of three animals (two animals with repetitive freezing and one animal with single freezing). During the further postcryotherapy observation period of 120 minutes, however, progressive and, finally, complete perfusion failure also occurred in all of these venules in the tumor margin. Thus, 2 hours after both single and repetitive cryotherapy, all microvessels of the tumors and the 3-mm zone of adjacent hepatic tissue showed a complete shutdown of perfusion.



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  		FIG. 6. Intravital fluorescence microscopy of the microvasculature of a CC531 colon tumor (TU) and the tumor-adjacent hepatic tissue (HT) at 2 weeks after tumor cell implantation under the capsule of the left liver lobe before (A) and 120 minutes after (B) cryothermia. Note the cryothermia-induced dilation of venules within the tumor margin (arrows and small inset in the lower part of the panel) and the complete shutdown of the microcirculation within the tumor, but also within the border zone of normal hepatic tissue. Contrast enhancement was achieved by sodium fluorescein 2 µmol/kg; original magnification, x 10 and x 20.

 


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  		FIG. 7. Venular diameters within the tumor center (A), the tumor margin (B), and the tumor-adjacent hepatic tissue (C) at 15, 60, and 120 minutes after single-freeze (closed circles) and double-freeze (open circles) cryothermia. Data are given as a percentage change from baseline (before cryothermia). Data are mean ± SEM; *P < .05 versus baseline.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated the successful development of a rat liver tumor model that allows intravital microscopic analysis of hepatic tumor vascularization and microvascular perfusion. With this model, the major finding of this study was that high-flow postcapillary venules in the tumor margin, which are associated with arteriolovenular shunting, critically determine the complete shutdown of microvascular perfusion, which can be achieved at approximately –35°C. In addition, we showed that repetitive freezing is not more effective for decreasing the tissue temperature at the tumor margin or for abolishing tumor microcirculation when compared with a single-freeze procedure.

Hepatic Tumor Microcirculation Model
A variety of different tumor models allow for intravital microscopic observations of the tumor microcirculation, including melanoma, glioma, and colon carcinoma.25–27 These tumor models, which are based on cell implantation into subcutaneous and striated muscle tissue of dorsal skinfold chambers of hamsters or nude and severe complete immune deficiency mice,28 demonstrate adequate microvascularization and tumor growth within 2 weeks after cell implantation. It is important to note that these tumors regularly show a higher capillary density in the proangiogenic tumor margin when compared with that of the tumor center and show an overall higher tumor capillary density when compared with that of the tumor-adjacent host tissue.26,29 The herein-presented model of CC531 colon carcinoma cell transplantation under the liver capsule also shows adequate tumor growth and microvascularization within 2 weeks after implantation; however, the capillary density of the tumor margin was only slightly (not significantly) higher than that of the tumor center, and the overall tumor capillary density was significantly lower when compared with the sinusoidal density of the tumor-adjacent liver tissue. The former may be explained by a somewhat delayed growth of the CC531 tumors, so that vascular regression within the tumor center is not initiated during the first 14 days. The latter is due to the fact that the liver is a highly vascularized organ with a sinusoidal density that is higher when compared with that of other tissues, such as subcutis or striated muscle.30

Strikingly, the CC531 tumors showed enlarged, high-flow postcapillary venules in the tumor margin, although the density of the venules was similar to that in the tumor center and the tumor-adjacent hepatic tissue. Because the density of the capillaries that drain into the venules was also not different between the tumor center and tumor margin and was even lower than the sinusoidal density in the adjacent hepatic tissue, the enlargement and high-flow conditions in the venules of the tumor margin can be due only to arteriolovenular shunting. This could indeed be confirmed by direct visualization by using intravital fluorescent microscopic techniques.

Cryotherapy-Associated Microvascular Perfusion Failure
Direct tumor cell destruction was considered as the primary mechanism of action of cryotherapy. As early as 1968, however, Gill et al.31 postulated that intracellular ice-crystal formation and enzyme degradation were not the only mechanisms of cryotherapy-induced cell injury. This was in line with other observations demonstrating that tumors implanted in rat livers were completely destroyed by cryotherapy without regrowth over weeks, whereas if the tumors were excised immediately after cryotherapy, a considerable number of cells were still viable and growing successfully in vitro in cell culture32 and in vivo after transplantation.33 Several early studies suggested that microvascular dysfunction may be one of these secondary factors that contribute to cryotherapy-induced cellular damage, probably by circulatory arrest and, thus, prolonged ischemia and anoxia.12,15,34 In 1990, Rubinsky et al.35 showed that the propagation of ice formation along the vascular system is associated with the destructive effects of cryotherapy; this further supported the view that microvascular dysfunction contributes to cryoinjury.13 Using indirect techniques such as laser Doppler fluxmetry14 but also direct imaging techniques such as intravital microscopy,16,36 recent studies confirmed in both cremaster muscle and liver preparations a complete shutdown of the microcirculation of normal tissue upon cryothermia.

A variety of previous studies attempted to determine the freeze temperature required for cell death; however, these studies demonstrated variable results that ranged between –20°C and –50°C as the critical temperature for irreversible cell damage.37–40 Others have criticized these studies because of the major drawback that they were performed in cell suspensions or cell layers and, thus, did not consider additional secondary effects that occur in perfused organ systems.12,13,15 In a recent study, we showed in normal rat liver (1) irreversible cessation of microvascular blood perfusion at 0°C initially after cryothermia and (2) progression of this ischemic border zone to approximately +8°C during the first 90 minutes after cryothermia.18 This resulted in complete necrosis after an 8-week observation period.16 However, different tissues may vary in response to distinct freeze temperatures, and tumors have been supposed to have a higher resistance to cold temperatures than healthy tissues.12,13,15,17,41 This study now demonstrates that in the center of liver tumors, a freeze temperature less than –50°C completely shuts down capillary and venular perfusion. In contrast, at the tumor margin, a freeze temperature of –32°C (single freezing) to –36°C (double freezing) initially completely shuts down only the capillary and small-venular perfusion, whereas cessation of blood flow in some larger venules is delayed. The fact that during 2 hours after cryotherapy perfusion is also completely abolished in the enlarged venules of the tumor margin indicates that these temperatures (–32°C to –36°C) may be critical but just sufficient to induce complete ischemia—and, thus, anoxia—to the marginal zone of the tumor. It is important to note that the freeze temperature of –30°C to 0°C that was applied to the tumor-adjacent liver tissue was able to completely abolish the perfusion in all microvascular segments directly after cryotherapy. This confirms the data of our previous studies16,18 and supports the view that healthy liver tissue is more susceptible than tumor tissue to low temperatures.

It is generally believed that ice formation in tissue can be divided into three grades: (1) rapid freezing in the center of the cryolesion adjoined by (2) a zone of moderate freezing and, finally, (3) slow freezing at the margin of ice formation. Cell survival seems to be superior at cooling rates of 10°C/minute,10,17 potentially leading to the highest rate of viable cells in the zone of moderate freezing. This is of clinical importance, because tumor recurrence is mostly observed in the tumor margin area,35 which corresponds to the cryotherapy zone of moderate freezing. More importantly, the proangiogenic tumor margin may be the critical target for cryothermia because of its special microvascular arrangements, which contain enlarged high-flow venules and arteriolovenular shunts. In this study, these vessels were the only ones that did not show a shutdown of perfusion directly after cryothermia, probably because of an impaired expansion of the cryotherapy zone by means of a heat-sink effect. This is in line with observations in nonmalignant tissue (cremaster muscle) which showed a short period of reperfusion after thawing only in large arterioles and venules but showed complete postfreeze reperfusion deficits in capillaries and postcapillary venules.36 In fact, under conditions of complete capillary stasis, blood flow in arterioles and venules can be maintained only in the presence of arteriolovenular shunts. Those arteriolovenular shunts could be directly visualized in this study in the tumor margin and have to be considered as the critical microvascular structures that determine the freeze temperature required to achieve complete shutdown of perfusion in the tumor microvasculature.

Most clinicians prefer the repetitive application of cryotherapy to achieve a more homogeneous freeze process and to avoid the undesirable heat-sink effect.1,11,31 There is evidence, however, that repetitive freezing is associated with a higher rate of complications and morbidity than single freezing.19,42 Therefore, the necessity of extremely low temperatures of less than –50°C all over the tumor tissue, which may be required to exert direct cell death, must be questioned. Cryotherapy-associated shutdown of the microcirculation may induce similar tumor destruction at more moderate temperatures of approximately –35°C and may thereby reduce the amount of injury to the tumor-adjacent healthy liver tissue.

In this study, freezing was performed until 0°C was achieved at 3 mm outside of the tumor margin. This ensured complete microvascular stasis in the normal liver tissue. By this, –32°C and –36°C were reached in the tumor margin by using the single- and repetitive-freeze procedures, respectively. Repetitive freezing did not double the effective freezing time compared with single freezing; this is because of an accelerated temperature decrease during the second freeze cycle.16,43 Within the tumor center and also within the tumor-adjacent hepatic tissue, single freezing was as effective as double freezing to directly shut down the perfusion in all microvascular segments. In the tumor margin, one of seven animals after single freezing and two of seven animals after double freezing showed temporary maintenance of large venular perfusion, which, however, ceased in all animals after a 2-hour postcryothermia observation period. Thus, in the model used herein, double freezing was not more effective compared with the single-freeze procedure in affecting tumor microcirculation.

In clinical practice, many tumors recur despite reaching extremely low temperatures. Because of the concern that recurrence is related to the heat-sink phenomenon, additional procedures, such as inflow occlusion or double-freeze modes, are applied. These procedures are thought to decrease the temperature and thus to increase the size of the iceball. Nonetheless, this still does not guarantee that all of the tumor tissue is exposed to a defined critical temperature. In accordance with Tranberg,44 we believe that the main problems with in situ ablation are the lack of good imaging techniques to determine the extent of disease and the lack of methods for real-time monitoring of tissue temperature. Future research should therefore focus on improving the imaging of the lesions and monitoring the temperature at the tumor margins. The results of our study may contribute to a more cautious but potentially successful clinical application of cryosurgery in that (1) they point toward the need for an accurate online monitoring system to measure the temperature at the tumor margin during the cryoablation procedure and that (2) the surgeons may aim at achieving a temperature of approximately –35°C at the tumor margin to guarantee complete shutdown of the microvasculature supplying the tumor tissue.

We conclude from this study (1) that there is no major difference between single and repetitive freezing in effectiveness in shutting down tumor micro-circulation, (2) that distinct hypervascularization within the tumor margin affords markedly lower freeze temperatures than are known for healthy liver tissue, and (3) that a tissue temperature of approximately –35°C is required to finally also abolish the perfusion of the large, high-flow tumor marginal venules, which participate in arteriolovenular shunting.


   ACKNOWLEDGMENTS
 
Supported by a grant of the Deutsche Fors-chungsgemeinschaft (Me 900/1–3 and 1–4).

Received for publication April 12, 2004. Accepted for publication November 29, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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