The Necrotizing Effect of Pulse Cryocycling on Liver Tissue FREE

Cryosurgery is an accepted method of treatment for various lesions.^1- 2 The efficacy of the treatment for tumors, however, is not yet considered to be equivalent to resection, because freezing was not supposed to achieve destruction of all tumor cells within the iceball.³ An important method that has been suggested to improve cryonecrosis is cryocycling, ie, the repeated freezing of the target tissue with intermittent thawing periods.^4- 5 The accepted method of cryocycling consists of 2 freezing-thawing periods, with the duration of each freezing period ranging from 3 to 15 minutes.^6- 7 The beneficial effect of cryocycling, however, has not been universally accepted.⁸ In the present study conducted on rabbits, we investigated the effect of pulse cryocycling (repeated, very short cycles of freezing and thawing) on normal liver tissue in vivo, compared with the standard method of cycling.

The study was performed on rabbits, under permit IL 38/08/2000 of the Committee for Experimental Studies on Animals of the Faculty of Medicine, Technion–Israel Institute of Technology, Haifa, Israel. A preliminary study was performed on 4 animals to determine the necessary period to obtain a definite histological picture of the cryolesion. The experiment itself was performed on a total of 18 female rabbits with a mean ± SD weight of 4402.8 ± 488.2 g (range, 3550-5300 g). They were divided into the following 3 groups of 6 animals each: control group (group 1, undergoing 5-minute cycles), group 2 (undergoing 15-second pulse cycles), and group 3 (undergoing 30-second pulse cycles) (Figure 1). The experiments were undertaken with the animals under general anesthesia. Induction was obtained by means of intravenous injection of 5 mg of diazepam and maintained with an intramuscular 2-mL injection of a combination of fentanyl citrate (0.315 mg/mL) and fluanisone (10 mg/mL) (Hypnorm; Janssen, Sounderton, England). During the entire operation, intravenous isotonic sodium chloride solution was administered at a rate of 0.7 mL/min. The core temperature, pulse, and blood pressure were monitored and recorded every 5 minutes immediately before onset of the first cryocycle and every 5 minutes thereafter for the whole period of the cryoprocess, with the aid of a Propaq apparatus (100 series; Protocol Systems Inc, Beaverton, Ore).

The liver was approached by means of a midline, upper abdominal incision. The left lateral lobe was isolated from the surrounding organs by means of a surgical glove pad to avoid freezing of adjacent organs. A 2-mm cryoprobe was inserted at a depth of 3 mm into the liver parenchyma. Freezing was performed with a Medical Cryohit 1 apparatus (Galil Medical, Upper Yokneam, Israel). This instrument uses argon and helium for freezing and thawing, respectively, based on the Joule-Thomson effect. When freezing is performed with argon, mainly the tip of the probe is affected and the loss of energy along the tubes is minimal. By inserting only the tip of the probe within the liver, we obtained half an iceball, the maximal diameter being on the surface of the liver. The protocol of the experiments is illustrated in Figure 1. In group 1, freezing was applied for 5 minutes, spontaneous thawing was allowed for 5 minutes, and the same cycles were repeated once more. In group 2, 10 repeated cycles of 15 seconds of freezing and 15 seconds of active thawing were performed for 5 minutes. After this period, spontaneous thawing was allowed for 5 minutes. The same procedure was repeated once more. Group 3 underwent a procedure similar to that of group 2, but the length of the cycles was 30 seconds (5 repeated cycles in each 5-minute freezing period). During the last freezing period of each experiment, the borders of the frozen tissue disk were marked on 2 opposite sides with the use of polypropylene monofilament 6-0 sutures (Ethicon Ltd, Edinburgh, Scotland) placed on opposite edges of the frozen disk, and ultrasonography, after inserting two 25-gauge needles at the border of the frozen lesion. The cryolesion was demonstrated by means of ultrasonographic examination, using an Acuson 128 XP scanner (Acuson, Mountain View, Calif) with a high-resolution 7-MHz linear transducer. The freeze front was visualized as a curvilinear hyperechoic rim, with posterior acoustic shadowing. Once the freezing process was completed, a needle was placed by the surgeon at each margin of the macroscopically visualized frozen lesion. On the ultrasonogram, the needles were detected as hyperechogenic vertical lines at the iceball edges, in complete correlation with the liver cryolesion size (Figure 2).

The diameter of the frozen lesion was measured at the end of the procedure, electronically on the ultrasonogram and metrically between the 2 marking sutures. The temperature of the cryolesion was measured at the end of the last freezing period using the thermocouple incorporated in the probe of the Medical Cryohit 1 apparatus, in its center with the probe performing the freezing and in its periphery. The later measurements were obtained using a second identical probe inserted at a depth of 3 mm, at 4 points on the border of the cryolesions, a quarter of a circle apart from each other. At the end of the operation, a subcutaneous injection of 150 mg of amoxicillin was administered.

The animals recovered and were killed on the following day with an intravenous injection of 300 mg of pentobarbital sodium and 5000 U of heparin sodium. The liver was harvested, and the diameter of the cryolesion was measured again. The timing of death on the day after the experiment was based on a pilot study in which the same experimental procedure was performed and the animals were killed on postoperative days 1, 3, 7, and 14.

The livers were immersed in 4% buffered formaldehyde solution, immediately on harvesting. After fixation, the tissue was cut into sections that included the cryolesion and the normal parenchyma at its periphery. Paraffin blocks were prepared from which 5-µm sections were cut, mounted on glass slides, and stained with hematoxylin-eosin. The diameter of the cryolesion was measured metrically twice more, before and after fixation with formaldehyde.

Unless otherwise indicated, data are given as mean ± SD. Comparison of 2 sets of data were performed using the t test for unpaired data, with the use of GraphPad InStat software V2.04 (GraphPad Software, San Diego, Calif). A P value of less than .05 was considered statistically significant.

No significant changes in blood pressure (Table 1) or pulse rates (Table 2) occurred during the cryoprocedures. The mean drop of the core temperature of the animals during the cryoprocedures was 1.05°C ± 0.51°C for group 1, 1.25°C ± 0.38°C for group 2, and 0.92°C ± 0.40°C for group 3. There was no statistically significant difference between any of the groups.

The mean minimal temperatures measured at the site of the cryoprobe (center of the lesion) during the cryoprocedures were −132.37°C ± 2.07°C for group 1, −129.83°C ± 1.47°C for group 2, and −131.50°C ± 1.23°C for group 3. There was no significant difference between the groups. The temperatures measured at the borders of the cryolesions in all 3 groups varied from 7°C to −1°C (mean, 2.88°C). During thawing, the temperatures measured by the cryoprobe (center of the cryolesion) and the probe positioned at the border of the lesion always exceeded 0°C, confirming complete thawing of the frozen tissue.

Ultrasonography showed a large acoustic shadow over the frozen tissue. The edges of the frozen tissue as detected by ultrasonography coincided with borders of the frozen lesion as visually marked by the needles (Figure 2).

The measurements of the cryolesions are given in Table 3. The apparent increase of the cryolesions, calculated by comparing the measurement after death with the size at the end of the cryoprocess (measurements A vs B in Table 3), was not statistically significant in any group. The apparent shrinkage in size of the lesion during the pathological fixation process was also not significant in any group (measurements B vs C in Table 3).

The microscopic examination invariably showed that already on the day after freezing of the liver, the resulting changes were obvious and clearly demarcated. The following 3 zones were seen in the histological sections (Figure 3): normal liver tissue at the periphery, necrotic liver tissue at the center, and an intermediate zone between the completely necrotic tissue at the center and the normal tissue at the periphery. The necrotic zone showed coagulation necrosis of the hepatocytes, the sinusoidal lining cells, and the portal tract cells. The intermediate zone was characterized by hepatocyte injury and preserved normal portal areas (Figure 4). The abnormal hepatocytes in the intermediate zone showed nuclear pyknosis, vacuolated cytoplasm, and blurred cell membrane. Neutrophils and nuclear cell debris accumulated in the border between central necrosis and the intermediate zone. The borders between the inner necrotic zone and the intermediate zone and those between the intermediate zone and the normal liver tissue were sharply delineated (Figure 4). The borders of the cryolesion, as visually marked during surgery and confirmed at harvest, coincided exactly with the border between normal liver tissue and the intermediate zone. The widths of the intermediate zones are listed in measurements E of Table 3. Although these were apparently thinner in group 3, there was no statistically significant difference among the 3 groups. The width of the intermediate zone relative to the diameter of the entire cryolesion was 4.4% ± 2.4% for group 1, 6.6% ± 1.3% for group 2, and 2.6% ± 0.6% for group 3. Although group 3 apparently attained the thinner intermediate zone, we found no statistically significant difference in the relative width of the intermediate zone between the pulse cryocycling groups compared with group 1. However, compared with group 3, group 2 attained a significantly thicker intermediate zone relative to the diameter of the entire lesion (P = .02).

Complete tumor resection is the best treatment of liver malignancies. However, in many cases, this is not feasible because of the advanced tumor stage, the need to sacrifice vital organs to achieve resection, or the poor general condition of the patient. To these patients, alternative methods of treatment, although yielding poorer results than surgery, are offered. One such therapeutic approach is cryosurgery, which refers to the in situ destruction of tumors using subzero temperatures. With the introduction of more maniable cryoapparatuses, the use of cryosurgery gained popularity.^3,6,9- 11

The mechanism of cell destruction by freezing is well documented and understood.^5,12 Cellular demise results from mechanical damage induced by intracellular ice crystal formation, dehydration, and ischemia due to circulatory arrest in the frozen tissue. It depends mainly on the temperature fall, cooling rate, and thawing velocity. These 3 mechanisms of cell destruction, however, are not equal throughout the iceball. One reason is the gradient of temperature within the iceball. This commonly increases by 10°C for each 1 mm further away from the center,¹⁰ with the lowest temperature being adjacent to the cryoprobe and temperatures reaching near 0°C at the border of the frozen tissue.^2,7 Another reason is the cooling rate,¹² which is faster at the center and slower in the periphery.⁵ Therefore, it was generally accepted that within the iceball, a central sphere of total cell destruction, an adjacent outer zone of partial cell destruction, and perhaps an outer zone of cell survival are found.² This concept and the observations that various tissues respond differently to cooling^5,13- 14 explain the claims of most authors that in clinical practice, an additional marginal layer of normal tissue should be incorporated within the iceball to ensure the complete inclusion of the tumor within the central sphere of total cellular destruction.^{2- 3,6,10,15- 16} Even with the observation of this safety margin, cryosurgery is considered only second best to surgical excision.^3,11

A major factor in the outcome of cryosurgery is the repeated use of freezing and thawing (cryocycling). The concept of cryocycling was based on several observations. It has been shown that cryocycling increases the size (area and volume) of frozen tissue,^7- 8,17 although opposite results have also been published.¹⁸ It has also been shown that by interrupting a given period of freezing, lower temperatures were obtained within the iceball in the last cycle compared with those achieved by 1 uninterrupted cryocycle of the same total duration. This is due to increased thermal conductivity of the tissues caused by the first period of freezing.^5,14 Dilley et al¹⁹ observed that single freezing obtained a mean 64.2% of necrosis from the original iceball volume, whereas double freezing increased it to 82.5%. Finally, it has been claimed that lethal intracellular ice is more likely to form with repeated cryocycles.²⁰

In cryocycling, the length of each freezing and thawing period is an important factor in determining the obtained degree of tissue damage. However, lengths of cycles used in the published data vary between 3 minutes⁷ and 15 minutes.^6,8 Gill et al¹⁷ have shown that the longer the cycle, the larger is the frozen volume of tissue, but the number of freezing episodes also affects the volume of frozen tissue. However, the effect of cryocycling on the range of total cell destruction within the iceball was not sufficiently documented, although it has been shown that cryocycling increased the diameter of complete necrosis within the iceball.¹⁹

The present study aimed to examine a different method of cryocycling, namely the substitution of the continuous freezing cycle by a score of very short periods of freezing, each followed by a similarly short phase of active thawing (pulse cryocycling). It also planned to examine whether pulse cryocycling could increase the radius of the inner sphere of total cell demise and reduce the thickness of the outer layer of incomplete cell destruction, which may possibly be a site for tumor recurrence after cryotherapy. Thus, reducing the width of the outer, incomplete cell destruction zone may have an impact on local recurrence rates.

An important factor in determining the size of the cryolesion is the intraoperative marking of its borders. Several methods have been used, including sutures (single or purse-string) or inserted cannulas, ink, and needles detected by means of ultrasound.^11,19 Ink marking is considered inaccurate because it spreads over or within the tissue, and no sharp marking can be obtained. Dilley et al¹⁹ considered sutures unreliable as well. However, they examined the lesions 1 month after cryosurgery. Ultrasonographical marking of the edges of the cryolesions was performed in several studies.^21- 23 In our study, we found an excellent correlation in the size of the cryolesions as measured both visually and sonographically. We also found that the visual appearance of the borders as marked with sutures corresponded to the margins observed microscopically. The difference between our results and those of others¹⁹ may be owing to the very short period in our study between placement of sutures and histological examination (1 day).

It has been observed that the iceball obtained by a single freezing period is larger than that obtained by the same freezing period when fragmented.¹⁸ In our study, 30-second pulses obtained slightly smaller lesions than regular cryocycling (control), but no statistical difference existed. However, the size of the lesions obtained by 15-second pulses were significantly smaller than those obtained by the control method. Our results concur with those of Lam et al.¹⁸ Thus, in respect to the size of the iceball, 30-second pulse cryocycling had no advantage over regular cryocycling. However, in respect to the size of the cryolesion and the width of the intermediate zone, the results of 15-second pulse cycling were inferior to those of both the standard and 30-second pulse cryocycling.

Results of histological examinations showed that freezing of normal liver tissue resulted in sharply delineated lesions.^{5,7- 8,10,14,19} At the borders of the cryolesion, a layer of neutrophils has been observed^8,24 up to 2 mm thick.⁸ We discerned a similar neutrophilic zone in all 3 groups. A marginal layer within the cryogenic lesion (2-5 mm thick) of partially damaged tissue has been reported.^2,5 In our study, the histological examination of the lesions obtained by each method of cycling showed total destruction of the liver cells up to the border of the neutrophilic layer. The outer border of this neutrophilic layer coincided with the margins of the cryolesions. These results concur with those of other investigators.⁸ In this respect, therefore, the present study did not show any advantage of pulse cryocycling compared with the control method of cycling. However, in tumor tissues, it appears that the margins of cryolesions are not crisp.^10,14 It is, therefore, possible that for other types of tissue, different methods of cycling may yield various results.

The temperature required to ensure total cellular necrosis was estimated to be as low as −40°C.² However, it was commonly stated in the cryosurgical literature that −20°C was lethal for cells and that this temperature should be produced to secure a destructive effect.⁵ The temperature at the edge of the cryolesion is higher than these values and was reported to range between 0°C⁷ and −6°C.² Our measurements showed a higher median temperature (2.88°C). However, because the thermocouple is at the tip of the probe, which is conically shaped and rather thick (2 mm), it might not have been placed exactly on the frozen border, making the measurements slightly inaccurate. In our study as well as in that of others,⁸ total hepatocellular demise was observed up to the borders of all cryolesions, ie, it has been achieved by subzero temperature higher than −20°C. This may be explained by the existence of factors in addition to the temperature fall, including the type of tissue submitted to freezing (it appears that normal liver is more susceptible to freezing than tumors), length and number of freezing periods, and speed of freezing and thawing, which determine cellular cryodestruction.

In our experimental model, total hepatocellular destruction was obtained up to the borders of the cryolesion by all cryocyling methods. The persistent presence of an intermediate zone of partial cell destruction within the borders of cryolesions, thin as it may be, is due to the different susceptibility of various histological structures to cryotherapy. Our findings also emphasize the need to exceed the limits of the target lesion during tumor cryotherapy by at least the width of the intermediate zone. The results achieved by 2 cycles of uninterrupted freezing (our control group) were similar to those achieved by 30-second pulse cryocycling, which did not provide any superiority in attaining cell demise within the cryolesion of normal liver tissue. Very short (15-second) pulse cryoablation proved unsatisfactory. However, one should not extrapolate these results to other types of tissues. Theoretically, in less susceptible tissues, 30-second pulse cryocycling may achieve better results than the currently used method of two 5-minute cryocycles. Further studies with tumor tissues are required.

Corresponding author: Moshe Hashmonai, MD, PO Box 359, Zikhron Ya'akov 30952, Israel (e-mail: hasmonai@inter.net.il).

Accepted for publication September 9, 2003.