Cryobiology 39, 262–270 (1999) Article ID cryo.1999.2209, available online at http://www.idealibrary.com on
Thyroid Cryotherapy in an Experimental Rat Model Lech Pomorski,* Magdalena Bartos,* Maria Matejkowska,† Maciej Amsolik,* and Krzysztof Kuzdak* *Clinic of Endocrinological and General Surgery, Institute of Endocrinology, Medical University of Lodz; and †Department of Pathomorphology, Copernicus Hospital of Lodz, 93-513 Lodz, Poland In recent years cryotherapy has been more and more frequently used for the treatment of tumors of different organs. Until now, the use of cryotherapy for the treatment of thyroid lesions, as well as histopathologic changes in thyroid tissue after cryotherapy, has not been described. Nitrous oxide cryotherapy of one thyroid lobe in twenty 12-week male Wistar rats was performed. After 2 and 4 weeks, the cryotreated thyroid lobe and the second lobe along with a part of the trachea, esophagus, and the subhyoid muscles adhering to the thyroid were excised and assessed macro- and microscopically. The macroscopic evaluation, performed 2 and 4 weeks postcryotherapy, revealed atrophy of the cryotreated lobe in 4 and 3 rats, respectively, and reduction of the cryotreated lobe dimensions in 6 and 7 rats, respectively. In the specimens of the lobes excised 2 weeks following cryotherapy, examined microscopically, necrosis, granulomatous inflammation, hemorrhages, and hemosiderin deposits were found most often, whereas in the specimens of the lobe excised after 4 weeks lymphocytic inflammation and fibrosis were mainly observed. No microscopic changes were observed in the thyroid lobes that were not frozen or in the parathyroid glands located inside these lobes or extrathyroidally, either ipsilaterally or contralaterally to the cryotreated thyroid lobes. There was no microscopic damage to other tissues adjacent to the thyroid gland. No rat developed vocal cord dysfunction after cryotherapy and no significant changes in serum calcium level before and after cryotherapy were observed. The results obtained show that it is possible to cryoblate thyroid tissue without damaging the tissues adjacent to the thyroid, as well as to spare function of the recurrent laryngeal nerves and parathyroid glands. © 1999 Academic Press Key Words: cryotherapy; thyroid pathology; thyroid treatment.
thyroid adenomas, respectively. In 1982, tetracycline injections into the cyst were suggested for the treatment of thyroid cysts (27), and in the next years the use of other sclerosing agents such as hydroxy-polietoksy-dodecaneScleroven (25), ethanol (26), and Paoscle (34) gave encouraging results for the treatment of thyroid nodules. In recent years cryotherapy has been more and more frequently used for the treatment of tumors of different organs such as lungs (3), liver (2, 18, 20, 21, 23, 28, 29, 32), breast (25), prostate (4 – 6, 9, 16, 24, 30), and larynx (13). Until now, according to data from the Medline database, the use of cryotherapy for the treatment of thyroid nodules has not been described and histopathologic changes in thyroid tissue after cryotherapy have not been reported. The aim of the study is to evaluate macroand microscopic changes in cryotreated normal rat thyroid tissue and the influence of thyroid cryotherapy on the function of the recurrent
The introduction of fine needle aspiration biopsy under ultrasound guidance for the diagnosis of solitary thyroid nodules has enabled the differentiation of benign and malignant thyroid lesions with high sensitivity and specificity. Meanwhile, researchers have noticed that less than 5% of patients operated on for nodular goiter had thyroid cancer (15, 17). This observation and the fact that thyroid surgery is not devoid of major complications such as injury to the recurrent laryngeal nerves and parathyroid glands have reduced the number of indications for operative treatment and have encouraged the application of new nonsurgical therapeutic methods. At present, hormonal treatment with thyroxine and radioiodine therapy are commonly used nonoperative treatment methods for benign, nontoxic thyroid nodules and hyperfunctional Received June 11, 1999; accepted October 1, 1999. This work was funded by institutional sources.
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Cryotherapy of the thyroid. 1. Probe tip of the cryosurgery device. 2. Thyroid lobe. 3. Trachea.
laryngeal nerves and parathyroid glands in an experimental rat model. Determining the changes resulting from cryoinjury in normal thyroid tissue is a necessary prerequisite to assessing the possible role of cryosurgery in treating abnormalities in thyroid tissue. MATERIAL AND METHODS
Experiments were performed on twenty 12week male Wistar rats (mean weight 155 6 12 g) in the Department of Experimental Surgery of the Clinic of Endocrinological and General Surgery of the Medical University of Lodz. An operation was performed under general anesthesia with ketamine given intramuscularly in a dose of 20 – 40 mg/kg body wt. During the operation the thyroid was dissected free. The longitudinal, transverse, and sagittal dimensions of the thyroid lobes ranged from 4.4 to 5.2 mm, from 3.2 to 3.9 mm, and from 2.8 to 3.3 mm, respectively (mean, 4.7 3 3.5 3 3 mm). Freezing was performed by means of a cryosurgery device (AK-1 ver. 2, constructed in the Institu-
tion of Medical Equipment of Warsaw), in which nitrous oxide, expanding rapidly through a small orifice at the sterile probe tip (Joule– Thompson effect) (14), was the source of freezing. When the device is operated, the temperature at the probe tip is 289°C. The right thyroid lobe was subjected to cryotherapy. The process of freezing was guided by visual monitoring and the iceball was allowed to extend within the thyroid lobe and 1 to 2 mm into the perithyroidal tissues. During the operation the freeze– thaw cycle was repeated twice. The time of the first freezing was 1 min and the second was 45 s. After each freezing, thyroid tissue was allowed to thaw at room temperature. The left thyroid lobe was not frozen (Fig. 1). The reoperation was performed under general anesthesia. The lobe after cryotherapy and the second lobe along with a part of the trachea, esophagus, and the subhyoid muscles adhering to the thyroid were removed after 2 weeks (in 10 rats) or 4 weeks (in 10 rats). The specimens
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obtained were immediately fixed in 10% formalin. The specimens were stained with hematoxylin and eosin and examined microscopically in the Department of Pathomorphology of our hospital. Before cryotherapy, just after the first operation and before the second one, the vocal cords were displayed on a monitor by means of a microlaparoscopic camera inserted into the rat throat to evaluate their function. Serum calcium concentrations were determined before cryotherapy, as well as 5 days and 2 weeks after it. RESULTS
The intraoperative macroscopic evaluation during the reoperation performed 2 and 4 weeks after cryotherapy revealed no remnants of the cryotreated right thyroid lobe (lobe atrophy) in four (40%) and three (30%) rats, respectively. Only fibrous tissue was seen on the surface of the trachea. In six (60%) and seven (70%) rats, respectively, reduction of the dimensions of the right cryotreated lobe (mean dimensions, 3.5 3 2 3 2 mm) was observed. No macroscopic lesions were found in the left thyroid lobe, which was not frozen, as well as in the tissues surrounding the thyroid gland. The microscopic evaluation of the paraffinembedded specimens of the lobes excised 2 weeks after cryotherapy revealed almost complete atrophy of the thyroid tissue in 4 (40%) rats (in which no remnants of the cryotreated lobe, except for fibrous tissue on the trachea surface, were found macroscopically) and a reduction in the thyroid tissue volume in the other 6 (60%) rats. In the thyroid tissue preserved, no areas of normal thyroid follicles were found. All thyroid cells were injured, and the following histopathologic findings were observed: necrosis (all 10 cases) (100%); hemorrhage (all 10 cases) (100%); hemosiderin deposits (9 cases) (90%); infiltration by granulocytes (8 cases) (80%) and lymphocytes (4 cases) (40%), fibrosis (4 cases) (40%); a reduction in the volume of the thyroid follicles (with a reduction in colloid volume inside the follicles) or lumen atrophy (all 10 cases) (100%) with simultaneous elon-
gation (cylindrical cells) (8 cases) (80%) or flattening (3 cases) (30%) of the cells of follicle epithelium; the disturbance of the thyroid tissue architecture along with cysts (4 cases) (40%). In many cases different histopathologic changes occurred together (Figs. 2 and 3). Necrosis, hemorrhage, hemosiderin deposits, granulocyte and lymphocyte infiltration, and fibrosis were to be found in nearly the whole cryoinjured thyroid lobe. Only in some peripheral parts of the lobe were thyroid follicles with reduced colloid volume or lumen atrophy and disturbances of the thyroid tissue architecture with cysts encountered. It was also revealed that 6 (60%) of the 10 rats had two or three parathyroid glands (in 4 and 2 rats, respectively) that were localized inside the cryotreated thyroid lobe. The microscopic evaluation of these parathyroid glands revealed necrosis, hemorrhage, and inflammatory infiltration. The microscopic evaluation of the specimens of the cryotreated lobes excised after 4 weeks following cryotherapy revealed almost complete atrophy of the thyroid tissue in 3 (30%) rats (in which no remnants of the cryotreated lobe were found macroscopically) and a reduction in the thyroid tissue volume in 7 (70%) rats. In the thyroid tissue preserved, no areas of normal thyroid follicles were found either. All thyroid cells were injured, and histopathologic changes included necrosis (all 10 cases) (100%); hemorrhage (9 cases) (90%); hemosiderin deposits (all 10 cases) (100%); infiltration by granulocytes (2 cases) (20%) and lymphocytes (9 cases) (90%); fibrosis (all 10 cases) (100%); adipose tissue (3 cases) (30%); a reduction in the volume of the thyroid follicles (with a reduction in colloid volume inside the follicles) or lumen atrophy (all 10 cases) (100%) with simultaneous elongation (cylindrical cells) (9 cases) (90%) or flattening (2 cases) (20%) of the cells of follicle epithelium; the disturbance of the thyroid tissue architecture along with the presence of cysts (8 cases) (80%). In many cases different histopathologic changes occurred together. Necrosis, hemorrhage, hemosiderin deposits, granulocyte and lymphocyte infiltration, and fibrosis involved
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FIG. 2. Thyroid lobe 2 weeks after cryotherapy (hematoxylin– eosin, 1003). 1. Granulocyte infiltration. 2, 3. Changes of the shape and volume of the thyroid follicles.
nearly the whole cryoinjured thyroid lobe. Only in some peripheral parts of the lobe were thyroid follicles with reduced colloid volume or lumen atrophy and disturbances of the thyroid tissue architecture with cysts encountered. It was also revealed that in 5 of 10 rats, two or three parathyroid glands (in 4 and 1 rat, respectively) were localized inside the cryotreated thyroid lobe. Their microscopic evaluation revealed necrosis and hemorrhage. No microscopic changes were observed in the left thyroid lobes, which were not frozen, or in the parathyroid glands located inside these lobes. No histologic changes were found in the parathyroid glands located extrathyroidally, either ipsilaterally or contralaterally to the cryotreated thyroid lobes (Fig. 4). There was no microscopic damage to the other tissues adjacent to the thyroid gland (the subhyoid muscles, trachea, esophagus, recurrent laryngeal nerves). No rat developed vocal cord dysfunction postcryotherapy. No significant changes in se-
rum calcium level before and after cryotherapy were observed either. DISCUSSION
Cryotherapy was first applied for therapeutic destruction of tissue in England between 1845 and 1851, when James Arnott used iced salt solutions of a temperature about 220°C to freeze advanced cancers in accessible sites, obtaining a reduction in tumor size and in pain (9). Initially, cryotherapy was used for the treatment of accessible lesions of skin and mucosa. In 1961 Cooper constructed a modern cryosurgical device, which enabled the use of cryosurgery for the treatment of visceral tumors, and he initiated many investigations to assess the usefulness of this method of treatment (14). At present, liquid nitrogen is most frequently used for cryotherapy, and the visceral tumors that are most often treated by cryotherapy are tumors of the liver (2, 18, 20, 21, 23, 28, 29, 32) and the prostate (4 – 6, 9, 16, 24, 30). In many
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FIG. 3. Inflammatory infiltration with fibrosis in the cryoablated thyroid lobe (hematoxylin– eosin, 1003).
centers satisfactory results were obtained by cryotherapy of inoperable prostate cancers, primary liver cancers, and metastases of large intestine cancers to the liver. However, until now, indications for this kind of treatment have not been determined and no long-term, randomized investigations comparing the results of cryotherapy and other treatment methods have been performed (2, 16, 20, 29, 30). Actual cell destruction requires temperatures of 220°C to 250°C (4, 12, 21). The temperature at the edge of the iceball is approximately 0°C, which is not lethal to the cells (6, 18). Therefore, it is essential for the iceball to extend a few millimeters beyond the margins of the zone that is to be destroyed. The freezing process in superficial lesions can be guided by visual monitoring, while that of deep lesions requires imaging techniques or temperature measurements (6, 14, 20, 29). In the past, thermocouples or tissue-impedance monitoring was applied to regulate the extent of freezing. However, both of these methods require direct im-
plantation of the monitoring probes into the tissue, and 1 mm error in the placement of a thermocouple or tissue-impendance probe can result in a difference in temperature measurement of 10 to 30°C (23). Currently, real-time ultrasound monitoring is most often used in probe placement and freezing (14). As visualization of the deeper parts of the ice formation can be impaired by acoustic reflection at the proximal edge of the iceball, other imaging techniques, including computed tomography or magnetic resonance, are advocated by several authors for monitoring the freezing process (32). It has recently been observed that after the tissue is frozen and allowed to thaw, the degree of destruction of tissue can be improved if it is frozen again; the second freezing travels through the tissue with an increased velocity, which ensures maximum destruction. Furthermore, it has been shown that cells which are viable after freezing are still susceptible to necrosis during thawing (19) and that rapid freezing and slow thawing are most lethal to cells
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FIG. 4. Normal parathyroid gland and normal thyroid tissue in the thyroid lobe located contralaterally to the cryotreated lobe.
(22). Meanwhile, two or more cycles of freeze– thaw do not result in more side effects (16, 18, 24, 33). Therefore, at present, the standard is to use at least two or three freeze–thaw cycles. During the experiment performed by us, after precise preparation, the thyroid was accessible and we could estimate the extent of freezing by relying on visual monitoring. Because the temperature at the edge of the iceball is about 0°C, i.e., higher than that needed for cell destruction (#220°C) (4, 12, 21), the iceball was allowed to extend into the perithyroidal tissues for a distance of approximately 1 to 2 mm. Based on the results of other authors, we conducted two freeze–thaw cycles to ensure maximum lethality. However, considering the fact that the freezing effect is more rapid during refreezing, we shortened the time of the second freezing in comparison with the first one (45 vs 60 s). Histopathologic changes which have been observed most often in other organs after cryotherapy are necrosis, inflammatory cell infiltra-
tion, hemosiderin deposition, granulation tissue, fibrosis, hyalinization, and calcification (4, 9, 31). Necrosis can be observed as early as a few hours following cryotherapy (8, 31), whereas fibrosis is usually seen after 4 – 6 weeks (8, 10, 22). In our experiment, therefore, microscopic evaluation of the thyroid tissue was performed 2 and 4 weeks after cryotherapy to compare the probable differences in histologic findings. In our material, we have also found some histopathologic changes which have been detected by other clinicians in different cryoablated tissues. In the specimens of the lobes excised 2 weeks after cryotherapy, necrosis, granulomatous inflammation, hemorrhages, and hemosiderin deposits were most often found, whereas in the specimens of the lobe excised after 4 weeks, lymphocytic inflammation and fibrosis were observed most commonly. It should be emphasized that we observed no areas of normal thyroid follicles in the cryoinjured thyroid lobe in any rat. The areas of necrosis were encountered
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in nearly the whole cryotreated thyroid lobe in all rats subjected to the experiment. Only in some peripheral parts of the lobe was no necrosis found, and the histologic changes included thyroid follicles with reduced colloid volume or lumen atrophy and disturbances of the thyroid tissue architecture with the presence of cysts. As the iceball was allowed to extend into the perithyroidal tissues for distances of only 1 or 2 mm, these sites of the lobe may represent areas at the edge of the freezing zone created by the cryoprobe that were probably not adequately frozen. We allowed the iceball to extend only 1–2 mm beyond the lobe margins in order to avoid damage to the tissues adjacent to the cryotreated thyroid lobe. Based on the results obtained it seems that it is essential either for the iceball to extend further beyond the margins of the cryotreated thyroid lobe or for more freeze–thaw cycles to be used. Several studies showed that cryoablated lesions could be left in places and that they would be resorbed, with a fibrous scar left behind in the area of cryotherapy (8, 10, 31). Resorption takes longer (several months) if larger tissue volumes are injured (8). In our study, almost complete atrophy of cryoinjured thyroid tissue was revealed 2 and 4 weeks following cryotherapy in four (40%) and three (30%) rats, respectively. In the other animals, the process of resolution was probably less intensive and the volume of cryoinjured thyroid tissue was larger. We did not observe either macroscopic or microscopic features of destruction in the tissues surrounding the thyroid gland, including the parathyroid glands located inside the thyroid lobe which was not subjected to freezing, or extrathyroidally, either contralaterally or ipsilaterally to the cryotreated lobe. Necrosis of the parathyroid glands was encountered only if the glands were located inside the frozen thyroid lobe. However, we noted no significant changes in serum calcium concentration before and after thyroid cryotherapy, which is an evidence of unaltered function of the parathyroid glands in which no microscopic changes were found. No histologic changes were seen in the recurrent laryngeal nerves either. No rat developed vocal
cord dysfunction after cryotherapy, which confirms unaltered function of the recurrent laryngeal nerves. Although our study only shows that a normal thyroid lobe can be destroyed by cryotherapy, one may expect, based on the positive results obtained after cryotherapy of the tumors of other organs, that diseased thyroid tissue can also be cryoablated. Further studies are needed to evaluate the influence of cryotherapy on focal thyroid lesions. However, one can expect that, like the tumors of other organs, thyroid tumors may be more resistant than the normal tissue to this treatment modality. A study by Bischof et al. (1), evaluating a cooling response in the normal tissues and tumor tissues, showed that at a given rate of cooling, tumor cells retain more water and are therefore more susceptible to dehydratation at a constant cooling rate, which results in their increased resistance to freezing. It was shown that blood vessels play some role in tumor destruction by cryotherapy. In a study by Neel et al. (22), tumors were implanted in the rat livers and then subjected to cryotherapy, with and without occlusion. Tumor ischemia, by virtue of eliminating the warming effect of blood, increased the effectiveness of cryotherapy. As many focal lesions, especially malignant ones, are better vascularized than normal thyroid tissue, one can, therefore, expect that their destruction will be more difficult. Early studies suggested that all living tissues subjected to temperature of or below 220°C for longer than 1 min would undergo necrosis (7). More recent investigations, however, have suggested that treatment of all areas of a malignant tumor to at least 240°C is necessary to minimize the likelihood of local recurrence and to eliminate errors in evaluation of actual tumor margins (11). Several studies have shown that in cryotreated prostate cancer patients viable tumor cells are most often found in the prostatic apex, the seminal vessels, and the gland base. These sites are the least accessible to cryoprobes. Therefore, it is recommended to use three or more freeze–thaw cycles to ensure complete destruction of these regions (9). It is difficult to assess whether the cryosensitivity of
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malignant thyroid tissue is similar to that of other tumor tissues, and further studies are required to estimate how many freeze–thaw cycles should be applied to cause its destruction. It seems that our results show that it is possible to cryoablate normal thyroid tissue without damaging the tissues adjacent to it and to spare function of the recurrent laryngeal nerves and parathyroid glands. These results, taken with those of other authors who have shown that it is possible to cryoablate tumor tissue in different organs, encourage the use of cryotherapy as a potential nonresectional treatment modality for localized lesions of the thyroid gland, both benign and possibly inoperable malignant ones. ACKNOWLEDGMENT The authors acknowledge the assistance of the Institution of Medical Equipment “Kriometrum” in the conducting of this research. REFERENCES 1. Bischof, J., Christov, K., and Rubinsky, B. A morphological study of cooling rate response in normal and neoplastic human liver tissue: Cryosurgical implications. Cryobiology 30, 482– 492 (1993). 2. Bismuth, H., and Fecteau, A. Kombinationstherapie in der Onkologie— das hepatocellula¨re Carcinom. Chirurg 69, 360 –365 (1998). 3. Bolliger, C. T. Introduction of different approaches to intrabronchial treatment. Monaldi. Arch. Chest. Dis. 51, 316 –324 (1996). 4. Borkowski, M., Robinson, M. J., Poppiti, R. J., Nash, S. C., and Arkadi, M. Histologic findings in postcryosurgical prostatic biopsies. Mod. Pathol. 9, 807– 811 (1996). 5. Carroll, P. R., Presti, J. C., Small, E., and Roach M., 3rd. Focal therapy for prostate cancer: Maximizing outcome. Urology 49 (Suppl. 3A), 84 –94 (1997). 6. Connolly, J. A., Shinohara, K., Presti, J. C., and Caroll P. R. Should cryosurgery be considered a therapeutic option in localized prostate cancer? Urol. Clin. N. Amer. 23, 623– 631 (1996). 7. Dow, J. A., and Waterhouse, K. An experimental study in lethal freezing temperatures of the prostate gland. J. Urol. 103, 454 – 457 (1970). 8. Dutta, P., Montes, M, and Gage, A. A. Experimental hepatic cryosurgery. Cryobiology 14, 598 – 608 (1977). 9. Falconieri, G., Lugnani, F., Zanconati, F., Signoretto, D., and Di-Bonito, L. Histopatology of the frozen prostate. The microscopic bases of prostatic carcinoma cryoablation. Pathol. Res. Pract. 192, 579 – 87 (1996).
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