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Laparoscopic cryosurgery of the kidney in the porcine model: an acute histological study
LAPAROSCOPIC CRYOSURGERY OF THE KIDNEY IN THE PORCINE MODEL: AN ACUTE HISTOLOGICAL STUDY STEPHEN Y. NAKADA, FRED T. LEE, JR, THOMAS WARNER, SUSAN G. CHOSY, AND TIMOTHY D. MOON
ABSTRACT Objectives. To verify histologically whether cryosurgery of the kidney can be accomplished reproducibly without injuring adjacent structures, using a combination of ultrasound and laparoscopic guidance. Materials and Methods. Six kidneys from three domestic female farm pigs were utilized in the study. Under general anesthesia and after obtaining pneumoperitoneum, the lower pole of the kidney was mobilized laparoscopically and the ureter and adjacent bowel were protected with saline-soaked gauze. Two 3.8 mm-cryoprobes were placed percutaneously into the lower pole and cryoablation was carried out under laparoscopic and ultrasound guidance using a double-freeze technique (10-minute freeze and 5-minute thaw cycles to a probe temperature of 2185°C to 2196°C) in five kidneys (one control). The kidneys, adjacent ureter and bowel were harvested acutely, and macroscopic, histologic, and electron microscopic evaluation of all specimens was performed. Results. Macroscopically, clear margins of cryodestruction corresponded with the ultrasound images of the iceball. In all five treated kidneys, reproducible cell death corresponding to visible margins of cryodestruction were verified histologically. Cell death was further corroborated by electron microscopy. Adjacent structures (ureter and bowel) were sectioned and no significant damage was noted. Blood pressure remained constant throughout the procedure. A crack in the renal parenchyma of one kidney was noted during the thaw phase; at harvest that animal was found to have an intraperitoneal hemorrhage. Conclusion. Combined laparoscopic and ultrasound-guided cryoablation of the lower pole of the kidney can be accomplished reproducibly in the porcine model without injury to adjacent structures. UROLOGY 51 (Suppl 5A): 161–166, 1998. © 1998, Elsevier Science Inc. All rights reserved.
A
braham T. K. Cockett, M.D., is known worldwide for his accomplishments in research; he has been a visionary in the global nature of urology. Less well known is what he has done for those fortunate enough to have been trained by him. While chairman at the University of Rochester, Dr. Cockett created a fertile environment for scholarly pursuits; he continues to provide constant support for all his former residents and fellows in academic urology. It is a great privilege to contribute to this Festschrift in his honor. The concept of cryosurgery of the kidney is not
From Departments of Surgery, Division of Urology(S.Y.N., T.D.M.), Radiology (F.T.L., S.G.C.), Pathology (T.W.) University of Wisconsin Medical School, Madison, Wisconsin This work was supported by a grant from the National Kidney Foundation of Wisconsin Reprint requests: Stephen Y. Nakada, M.D., University of Wisconsin Medical School, Department of Surgery- Division of Urology, G5/343 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792 © 1998, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
novel. Onik and colleagues reported accurate correlation between ultrasound characteristics of the iceball and cell death while performing open renal cryosurgery in dogs.1 Other investigators have attempted cryosurgery of the kidney in other animal models and in human subjects via both open laparotomy and percutaneously.2– 4 Recent reports of laparoscopic radical nephrectomy have created great interest in the field of minimally invasive renal cancer therapy.5 However, to date laparoscopic radical nephrectomy remains a technically challenging procedure, to be performed by more experienced urologic laparoscopists. While open cryosurgery of the kidney has been reported,2 laparoscopic cryosurgery of the kidney would offer a more straightforward minimally invasive treatment for select renal tumors. In addition, laparoscopic access to the site of cryoablation has several potential benefits over percutaneous access. These include visualization of the margins of cryoablation, shielding of adjacent structures, and 0090-4295/98/$19.00 PII S0090-4295(98)00080-6 161
potential control of hemorrhage. Herein we present an acute histologic study of laparoscopic ultrasound– guided cryorenal ablation in the porcine model. MATERIALS AND METHODS Three domestic female farm pigs were utilized for the study; two animals underwent bilateral lower pole cryoablation, and one underwent unilateral cryosurgery and a sham procedure.
SURGICAL PROCEDURE Anesthesia was initiated using a combination of ketamine (35 mg/kg IM), xylazine (5 mg/kg IM), and butorphenol (0.1 mg/kg IM). The pig was intubated and anesthesia maintained by inhaled halothane. In the supine position, a standard femoral cutdown was performed in the left groin, and a 5F angiographic catheter advanced into the aorta for blood pressure monitoring. Using a Veress needle, pneumoperitoneum was established and a 12-mm trocar was inserted. The pig was placed in the lateral decubitis position and two more 12-mm trocars were inserted into the midaxillary line (MAL) above and below the level of the umbilicus. The colon was mobilized, and the lower pole of the kidney dissected free. Saline-soaked surgical sponges were placed around the kidney to help prevent inadvertent damage to adjacent visceral structures. Next, two 3.8-mm cryoprobes (Cryomedical Sciences, Rockville, MD) were inserted percutaneously under ultrasound guidance into the exposed lower pole of the kidney (Fig. 1). This was accomplished by initially passing an 18-gauge Teflon-coated diamond-tipped needle percutaneously into the lower pole, and advancing a 0.035inch curved guidewire through the needle. Next, a 10F dilating system and sheath (Cook Urological, Spencer, IN) was inserted percutaneously over the guidewire. The guidance system and cryoprobes were positioned into the lower pole utilizing a combination of ultrasound (5.0 mHz curvilinear transducer, SSR 2000, Aloka, Wallingford, CT) and laparoscopic guidance (Olympus America, Mellville, NY). A double-freeze technique (10-minute freeze and 5-minute thaw cycles)6 was utilized to freeze the lower pole under ultrasound guidance (Table I, Fig. 2). Target temperatures for all probes were 2185°C to 2196°C. The blood pressure and heart rate were monitored and recorded throughout the procedure via arterial line. Animals were euthanized using an overdose of Beuthanasia-D intravenously and kidneys harvested after reduction of the pneumoperitoneum and closure of the ports. Harvesting of the kidneys was performed through a midline incision and adjacent ureter and bowel were sampled and studied (vide infra).
FIGURE 1. Schematic of kidney and collecting system with two 3.8-mm outer-diameter cryoprobes aligned in the lower pole and subsequent iceball.
GROSS AND MICROSCOPIC PATHOLOGY The abdominal cavity was explored to rule out significant damage to other organs. Kidneys were removed en bloc and sectioned into multiple 3- to 5-mm slices in the coronal plane for gross inspection. The size of the cryolesion, completeness of ablation of the lower pole, evidence of complications and viability of the remainder of the kidney were assessed. The adjacent ureter and bowel were also sampled and studied histologically. The kidneys were then fixed in 10% neutral buffered formalin. Tissue blocks taken after photography and mapping of affected areas were refixed in formalin. All areas were sampled, including hilar blood vessels, lymph nodes, renal pelvis and ureter. These were embedded in paraffin, sectioned at 5 mm and stained with hematoxylin– eosin. The TUNEL technique7 was used to identify apoptotic nuclei. In this technique, the nick ends of DNA fragmented by the spe162
FIGURE 2. Longitudinal ultrasound image showing iceball covering lower pole parenchyma (see label). Note preserved upper pole (see label). Iceball causes complete reflection of the ultrasound beam with posterior acoustic shadowing.
cific endonucleases activated during apoptosis are labeled with biotinylated dUTP using terminal deoxynucleotidyl transferase. The end product is detected by streptavidin– horseradish peroxidase chromogen generation. Small blocks of tissue were fixed in 2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate, postfixed in 1% osmium tetroxide, dehydrated in graded alcohols and propylene oxide, embedded in UROLOGY 51 (Supplement 5A), May 1998
TABLE I. Pig
Kidney
Laparoscopic cryosurgery of the kidney: operative and macroscopic data
OR Time (minutes)
Time of Freeze/Thaw Cycle (minutes)
Temperature of Probe (°C)
Time of Harvest
Kidney Weight (g)
Lesion Weight (g)
Macroscopic Findings Normal parenchyma; clear margin of hemorrhagic necrosis; hematoma; cracked parenchyma Clear margin of hemorrhagic necrosis, no hematoma; intact parenchyma Clear margin of hemorrhagic necrosis; no hematoma; intact parenchyma
1
Left Right
90
NA 35
NA –185 to –196
100 minutes
47.9 55.6
NA 24.3
2
Left Right
122
28 53
–185 to –196 –185 to –196
Immediate Immediate
59.2 63.8
22.1 10.7
3
Left Right
130
33 35
–185 to –196 –185 to –196
Immediate
65.9 79.4
18.6 24.3
araldite, and thin-sectioned. The sections were stained with uranyl acetate and lead citrate and examined in a Hitachi H-500 electron microscope.
OPERATIVE RESULTS AND MACROSCOPIC ANALYSIS The total procedure time (from incision to closure) was 90 minutes for pig 1 (unilateral cryosurgery and sham), 122 minutes for pig 2, and 130 minutes for pig 3 including time for the freeze and thaw cycles (Table I). Blood pressure remained stable throughout the procedure in all cases. No intraoperative complications were noted aside from one case in which the renal parenchyma had cracked following thaw (pig 1); significant hemorrhage was noted in the abdomen at the time of harvest. Harvest was immediate in all cases but one, where harvest was performed 100 minutes after skin closure. At harvest, the adjacent viscera appeared intact macroscopically, and each kidney had distinct demarcation of hemorrhagic necrosis which did not follow an anatomic line of cleavage (Fig. 3). There was some hemorrhage into the renal sinus and the calyces and beneath the renal capsule. Kidney weights ranged from 55.6 to 79.4 g for treated kidneys, and lesion weights (following excision by T.W.) ranged from 10.7 to 24.3 g (Table I). During the thaw phase, an increase in the pneumoperitoneum was noted in each case; this was controlled by venting the ports.
es; these may have been situated at the advancing edge of the freezing zone. Many glomeruli were hemorrhagic, and some showed early necrotic changes; fibrin deposition was seen in capillaries and in some interlobular arteries and veins. The hemorrhage extended into the pericaliceal fat. The caliceal, pelvic and ureteric urothelium was intact over the papillae which were not affected by freezing. The urothelium over damaged papillae was almost completely exfoliated; when half of the papilla was damaged, there was corresponding loss of the associated urothelium with sparing of urothelium over intact portions of the papilla. There was focal loss of ureteric epithelium but this was possibly artifactual in origin due to incomplete fixation. Intramural hemorrhage was seen in some large arteries. Adjacent normal renal tissue was clearly demarcated from the hemorrhagic cryoablated areas and the microscopic features were consistent with cell death. Tissue from the control kidney also demonstrated normal renal tubules and glomeruli. The inflammatory response was minimal and consisted of mild focal interstitial polymorphonuclear leukocytes (PMN) infiltration; PMNs were also seen in the endothelium of some blood vessels. Margination of nuclear chromatin resembled the change seen in apoptotic cells. However, the results of the TUNEL technique were negative in these cells.
LIGHT MICROSCOPY Well-defined hemorrhagic zones were present (Fig. 4) in the lower pole of each kidney along with subcapsular hemorrhage. The renal tubules showed early necrotic changes with ill-defined cell borders (Fig. 5) and epithelial exfoliation. Nuclei were vesicular and margination of chromatin was identified in many areas. In some papillae, several terminal collecting ducts showed minimal chang-
ELECTRON MICROSCOPY Ultrastructural examination of kidney from frozen areas showed swelling of tubular epithelial cells. The mitochondria were swollen and cristae destroyed. Many luminal microvilli showed swelling and a number of luminal cells were disrupted. Basal laminae were intact, while the nuclei showed variable degrees of margination of chromatin (Fig.
RESULTS
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163
FIGURE 3. Hemorrhagic appearance of cryolesion in the lower pole of the kidney. Note clear line of demarcation in the renal parenchyma.
FIGURE 5. Detail of renal tubules showing interstitial hemorrhage, ill-defined borders of cells and margination of nuclear chromatin (arrows) (Hematoxilyn and Eosin 400X).
6). These changes were of such a degree that cell destruction was irreversible. COMMENT The “gold standard” for renal cell carcinoma in patients with a normal contralateral kidney has been radical nephrectomy. Recently, acceptable results have been reported in two large series by major centers utilizing open partial nephrectomy.8,9 Although initially adopted as the procedure of choice in patients with functionally solitary kidneys, the procedure has received acceptance as an option for smaller (,5 cm) polar malignancies. This experience has arisen also in part from the five-fold increase in detection of small (,3 cm) renal tumors between 1974 –1977 and 1982–1985 as a result of more modern cross-sectional imaging modalities.10 Licht and Novick found no signifi164
FIGURE 4. Clearly defined line of demarcation between lesion and intact renal parenchyma (hematoxylin and eosin, 340).
FIGURE 6. Electron micrograph of cryolesion showing pale degenerating cells (D), ill-defined cell borders (arrow) degenerating mitochondria (arrowheads), and marginated nuclear chromatin (M) (X 3,900).
cant differences in survival using this renal-sparing approach, as compared with conventional radical nephrectomy.8 The first laparoscopic nephrectomy was reported by Clayman et al. in 1991.11 Since then, the benefits of shortened hospital stay and convalescence have been well described; yet for most urologists, despite technical innovations, the procedure continues to be more time consuming and technically demanding than its open counterpart. The learning curve is further complicated by the relative rarity of benign nephrectomies. Laparoscopic partial nephrectomy was first reported in 1993 when Winfield et al. performed a laparoscopic partial nephrectomy for a benign, infected kidney.12 While possible, the authors noted that the technical demands and prolonged operative time inherent in this procedure made it somewhat problematic. UROLOGY 51 (Supplement 5A), May 1998
More recently, McDougall et al. reported experience with laparoscopic radical nephrectomy, demonstrating the feasibility of laparoscopic surgery in extirpative renal oncologic surgery.5 Tumors as large as 14 cm have been removed via the laparoscope in an impermeable entrapment sack with low morbidity and without tumor recurrence, albeit at early follow-up.5 In parallel to these strides in laparoscopic urology, the use of percutaneous, ultrasound-guided cryosurgery as a potentially curative procedure for carcinoma of the prostate was reported in 1993.13 Although the data were preliminary, a reproducible result was achievable with ultrasound guidance. Clinical data have continued to accumulate that support cryosurgery as a potential minimally invasive option in locally confined prostate cancer.14 Work has shown that complete ablation of prostatic tissue in dogs is achievable at the microscopic level when using thermosensor control.6 However, reports of severe complications with cryosurgery of the prostate, including urethrorectal fistula have slowed the widespread use of the technique.15 Cryosurgery of the kidney has distinct anatomic advantages over cryosurgery of the prostate. The kidney is surrounded by fat; the kidney may also be freed easily from surrounding structures, unlike the prostate. Similarly, the spherical configuration of many renal tumors follows the geometry of the iceball from a single probe. In 1993, Onik et al. reported the use of ultrasound-guided cryosurgery of the kidney via open laparotomy in the dog.1 This study demonstrated the feasibility of the technique, as well as the ability to monitor the cryolesion with ultrasound. Subsequently, Uchida and colleagues did a descriptive study using percutaneous cryosurgery in dogs and in two human subjects.4 This study showed the increased efficacy of tumor kill in a model of the unperfused kidney due to lack of rapid rewarming from circulating blood. Both patients died from pre-existing metastatic disease. Delworth et al. reported cryosurgery of a midpole renal cell carcinoma in a patient with a solitary kidney via open laparotomy, with success at 1-month follow-up.3 Questions have been raised as to subsequent bleeding with this approach, and the efficacy of tumor kill. Similarly, the ability to maintain adequate surgical margins while controlling damage to surrounding structures remains histologically unproven. This acute study verifies that cell death can be reproducibly and effectively achieved with laparoscopic cryosurgery of the kidney without significant injury to adjacent structures. Overall, the microscopic appearance of these cryolesions demonstrated that the damage was severe and irreversible even though the interval between rewarming of UROLOGY 51 (Supplement 5A), May 1998
tissue and harvesting the kidneys was brief (0 –100 minutes). What remains unclear is the temperature required to achieve adequate cryoablation of tissue and how best to monitor the temperature at the margins of the iceball. In addition, the question arises whether the entire procedure can be carried out percutaneously; Cozzi et al. reported 5 of 10 animals treated with cryoablation had subsequent hemorrhage following thaw.3 Our experience was consistent with this observation. Laparoscopic access to the cryotherapy site may allow for control of minor hemorrhage using avitene pledgets and laparoscopic suturing techniques. Other concerns include damage to adjacent structures, adequacy of tumor staging, delayed hemorrhage, and abscess formation. Further studies are ongoing in this regard. Successful laparoscopic cryosurgery of select polar renal tumors should result in shortened operative times and hospital stays and the ability to perform a curative operation in patients with significant comorbid illnesses as compared with more traditional therapies. The availability of cryosurgical probes for the laparoscope, intraoperative and laparoscopic ultrasound transducers, and sophisticated techniques of selective arterial embolization further increase the potential for clinical application of laparoscopic cryosurgery of the kidney. CONCLUSION This study has evaluated histologically the safety and efficacy of transperitoneal laparoscopic cryoablation of the kidney in the porcine model. Its effectiveness in controlling tumor progression as well as long-term adverse effects, including delayed bleeding and abscess formation, remain untested. We believe, if clinically feasible and effective, laparoscopic cryosurgery of the kidney would provide a simple, minimally invasive technique with which to treat selected renal tumors. ACKNOWLEDGMENTS. We thank Vida Stark for the TUNEL preparation. REFERENCES 1. Onik GM, Reyes G, Cohen JK, and Porterfield B: Ultrasound characteristics of renal cryosurgery. Urology 42: 212– 215, 1993. 2. Cozzi PJ, Lynch WJ, Collins S, Vonthethoff L, and Morris DL. Renal cryotherapy in a sheep model: a feasibilty study. J Urol, 157: 710 –712, 1997. 3. Delworth MG, Pisters LL, Fornage BD, and von Eschenbach, AC: Cryotherapy for renal cell carcinoma and angiomyolipoma. J Urol 155: 252–255, 1996. 4. Uchida M, Imaide Y, Sugimoto K, Uehara H, and Watanabe H: Percutaneous cryosurgery for renal tumors. Br J Urol 75: 132–137, 1995. 5. McDougall EM, Clayman RV, and Elashry OM: Laparoscopic radical nephrectomy for renal tumor: the Washington University experience. J Urol 155: 1180 –1185, 1996. 165
6. Kryger JV, Chinn DO, Messing EM, Siders DB, Lee FT Jr, Zarvan NP, Lee F. Cryosurgical ablation of the prostate in a canine model. Presented at the North Central Section Meeting of the American Urology Association, Minneapolis, 1995. 7. Gavrieli Y, Sherman Y, and Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493–501, 1992. 8. Licht MR, and Novick AC: Nephron sparing surgery for renal cell carcinoma. J Urol 149: 1–7, 1993. 9. Lerner SE, Hawkins CA, Blute ML, Grabner A, Wollan PC, Eickholt JT, and Zincke H: Disease outcome in patients with low stage renal cell carcinoma treated with nephron sparing or radical surgery. J Urol 155: 1868 –1873, 1996. 10. Smith SJ, Bosniak MA, Megibow AJ, Hulnick DH, Horii SC, and Raghavendra BN: Renal cell carcinoma: earlier discovery and increased detection. Radiology 170: 699 –703, 1989.
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11. Clayman RV, Kavoussi LR, Soper NJ, Dierks SM, Meretyk S, Darcy MD, Roemer FD, Pingleton ED, Thomson PG, and Long SR: Laparoscopic nephrectomy: Initial case report. J Urol 146: 278 –282, 1991. 12. Winfield HN, Donovan JF, Godet AS, and Clayman RV: Laparoscopic partial nephrectomy: Initial case report for benign disease. J Endourol 7: 521–526, 1993. 13. Onik GM, Cohen JK, Reyes GD, Rubinsky B, Chang Z, and Baust J: Transrectal ultrasound-guided percutaneous radical cryosurgical ablation of the prostate. Cancer 72: 1291– 1299, 1993. 14. Shinohara K, and Carroll PR: Improved results of cryosurgical ablation of the prostate. J Urol 153: 385A, 1995. 15. Cox RL, and Crawford ED: Complications of cryosurgical ablation of the prostate to treat localized adenocarcinoma of the prostate. Urology 45:932-935, 1996.
UROLOGY 51 (Supplement 5A), May 1998
ABSTRACT Objectives. To verify histologically whether cryosurgery of the kidney can be accomplished reproducibly without injuring adjacent structures, using a combination of ultrasound and laparoscopic guidance. Materials and Methods. Six kidneys from three domestic female farm pigs were utilized in the study. Under general anesthesia and after obtaining pneumoperitoneum, the lower pole of the kidney was mobilized laparoscopically and the ureter and adjacent bowel were protected with saline-soaked gauze. Two 3.8 mm-cryoprobes were placed percutaneously into the lower pole and cryoablation was carried out under laparoscopic and ultrasound guidance using a double-freeze technique (10-minute freeze and 5-minute thaw cycles to a probe temperature of 2185°C to 2196°C) in five kidneys (one control). The kidneys, adjacent ureter and bowel were harvested acutely, and macroscopic, histologic, and electron microscopic evaluation of all specimens was performed. Results. Macroscopically, clear margins of cryodestruction corresponded with the ultrasound images of the iceball. In all five treated kidneys, reproducible cell death corresponding to visible margins of cryodestruction were verified histologically. Cell death was further corroborated by electron microscopy. Adjacent structures (ureter and bowel) were sectioned and no significant damage was noted. Blood pressure remained constant throughout the procedure. A crack in the renal parenchyma of one kidney was noted during the thaw phase; at harvest that animal was found to have an intraperitoneal hemorrhage. Conclusion. Combined laparoscopic and ultrasound-guided cryoablation of the lower pole of the kidney can be accomplished reproducibly in the porcine model without injury to adjacent structures. UROLOGY 51 (Suppl 5A): 161–166, 1998. © 1998, Elsevier Science Inc. All rights reserved.
A
braham T. K. Cockett, M.D., is known worldwide for his accomplishments in research; he has been a visionary in the global nature of urology. Less well known is what he has done for those fortunate enough to have been trained by him. While chairman at the University of Rochester, Dr. Cockett created a fertile environment for scholarly pursuits; he continues to provide constant support for all his former residents and fellows in academic urology. It is a great privilege to contribute to this Festschrift in his honor. The concept of cryosurgery of the kidney is not
From Departments of Surgery, Division of Urology(S.Y.N., T.D.M.), Radiology (F.T.L., S.G.C.), Pathology (T.W.) University of Wisconsin Medical School, Madison, Wisconsin This work was supported by a grant from the National Kidney Foundation of Wisconsin Reprint requests: Stephen Y. Nakada, M.D., University of Wisconsin Medical School, Department of Surgery- Division of Urology, G5/343 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792 © 1998, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
novel. Onik and colleagues reported accurate correlation between ultrasound characteristics of the iceball and cell death while performing open renal cryosurgery in dogs.1 Other investigators have attempted cryosurgery of the kidney in other animal models and in human subjects via both open laparotomy and percutaneously.2– 4 Recent reports of laparoscopic radical nephrectomy have created great interest in the field of minimally invasive renal cancer therapy.5 However, to date laparoscopic radical nephrectomy remains a technically challenging procedure, to be performed by more experienced urologic laparoscopists. While open cryosurgery of the kidney has been reported,2 laparoscopic cryosurgery of the kidney would offer a more straightforward minimally invasive treatment for select renal tumors. In addition, laparoscopic access to the site of cryoablation has several potential benefits over percutaneous access. These include visualization of the margins of cryoablation, shielding of adjacent structures, and 0090-4295/98/$19.00 PII S0090-4295(98)00080-6 161
potential control of hemorrhage. Herein we present an acute histologic study of laparoscopic ultrasound– guided cryorenal ablation in the porcine model. MATERIALS AND METHODS Three domestic female farm pigs were utilized for the study; two animals underwent bilateral lower pole cryoablation, and one underwent unilateral cryosurgery and a sham procedure.
SURGICAL PROCEDURE Anesthesia was initiated using a combination of ketamine (35 mg/kg IM), xylazine (5 mg/kg IM), and butorphenol (0.1 mg/kg IM). The pig was intubated and anesthesia maintained by inhaled halothane. In the supine position, a standard femoral cutdown was performed in the left groin, and a 5F angiographic catheter advanced into the aorta for blood pressure monitoring. Using a Veress needle, pneumoperitoneum was established and a 12-mm trocar was inserted. The pig was placed in the lateral decubitis position and two more 12-mm trocars were inserted into the midaxillary line (MAL) above and below the level of the umbilicus. The colon was mobilized, and the lower pole of the kidney dissected free. Saline-soaked surgical sponges were placed around the kidney to help prevent inadvertent damage to adjacent visceral structures. Next, two 3.8-mm cryoprobes (Cryomedical Sciences, Rockville, MD) were inserted percutaneously under ultrasound guidance into the exposed lower pole of the kidney (Fig. 1). This was accomplished by initially passing an 18-gauge Teflon-coated diamond-tipped needle percutaneously into the lower pole, and advancing a 0.035inch curved guidewire through the needle. Next, a 10F dilating system and sheath (Cook Urological, Spencer, IN) was inserted percutaneously over the guidewire. The guidance system and cryoprobes were positioned into the lower pole utilizing a combination of ultrasound (5.0 mHz curvilinear transducer, SSR 2000, Aloka, Wallingford, CT) and laparoscopic guidance (Olympus America, Mellville, NY). A double-freeze technique (10-minute freeze and 5-minute thaw cycles)6 was utilized to freeze the lower pole under ultrasound guidance (Table I, Fig. 2). Target temperatures for all probes were 2185°C to 2196°C. The blood pressure and heart rate were monitored and recorded throughout the procedure via arterial line. Animals were euthanized using an overdose of Beuthanasia-D intravenously and kidneys harvested after reduction of the pneumoperitoneum and closure of the ports. Harvesting of the kidneys was performed through a midline incision and adjacent ureter and bowel were sampled and studied (vide infra).
FIGURE 1. Schematic of kidney and collecting system with two 3.8-mm outer-diameter cryoprobes aligned in the lower pole and subsequent iceball.
GROSS AND MICROSCOPIC PATHOLOGY The abdominal cavity was explored to rule out significant damage to other organs. Kidneys were removed en bloc and sectioned into multiple 3- to 5-mm slices in the coronal plane for gross inspection. The size of the cryolesion, completeness of ablation of the lower pole, evidence of complications and viability of the remainder of the kidney were assessed. The adjacent ureter and bowel were also sampled and studied histologically. The kidneys were then fixed in 10% neutral buffered formalin. Tissue blocks taken after photography and mapping of affected areas were refixed in formalin. All areas were sampled, including hilar blood vessels, lymph nodes, renal pelvis and ureter. These were embedded in paraffin, sectioned at 5 mm and stained with hematoxylin– eosin. The TUNEL technique7 was used to identify apoptotic nuclei. In this technique, the nick ends of DNA fragmented by the spe162
FIGURE 2. Longitudinal ultrasound image showing iceball covering lower pole parenchyma (see label). Note preserved upper pole (see label). Iceball causes complete reflection of the ultrasound beam with posterior acoustic shadowing.
cific endonucleases activated during apoptosis are labeled with biotinylated dUTP using terminal deoxynucleotidyl transferase. The end product is detected by streptavidin– horseradish peroxidase chromogen generation. Small blocks of tissue were fixed in 2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate, postfixed in 1% osmium tetroxide, dehydrated in graded alcohols and propylene oxide, embedded in UROLOGY 51 (Supplement 5A), May 1998
TABLE I. Pig
Kidney
Laparoscopic cryosurgery of the kidney: operative and macroscopic data
OR Time (minutes)
Time of Freeze/Thaw Cycle (minutes)
Temperature of Probe (°C)
Time of Harvest
Kidney Weight (g)
Lesion Weight (g)
Macroscopic Findings Normal parenchyma; clear margin of hemorrhagic necrosis; hematoma; cracked parenchyma Clear margin of hemorrhagic necrosis, no hematoma; intact parenchyma Clear margin of hemorrhagic necrosis; no hematoma; intact parenchyma
1
Left Right
90
NA 35
NA –185 to –196
100 minutes
47.9 55.6
NA 24.3
2
Left Right
122
28 53
–185 to –196 –185 to –196
Immediate Immediate
59.2 63.8
22.1 10.7
3
Left Right
130
33 35
–185 to –196 –185 to –196
Immediate
65.9 79.4
18.6 24.3
araldite, and thin-sectioned. The sections were stained with uranyl acetate and lead citrate and examined in a Hitachi H-500 electron microscope.
OPERATIVE RESULTS AND MACROSCOPIC ANALYSIS The total procedure time (from incision to closure) was 90 minutes for pig 1 (unilateral cryosurgery and sham), 122 minutes for pig 2, and 130 minutes for pig 3 including time for the freeze and thaw cycles (Table I). Blood pressure remained stable throughout the procedure in all cases. No intraoperative complications were noted aside from one case in which the renal parenchyma had cracked following thaw (pig 1); significant hemorrhage was noted in the abdomen at the time of harvest. Harvest was immediate in all cases but one, where harvest was performed 100 minutes after skin closure. At harvest, the adjacent viscera appeared intact macroscopically, and each kidney had distinct demarcation of hemorrhagic necrosis which did not follow an anatomic line of cleavage (Fig. 3). There was some hemorrhage into the renal sinus and the calyces and beneath the renal capsule. Kidney weights ranged from 55.6 to 79.4 g for treated kidneys, and lesion weights (following excision by T.W.) ranged from 10.7 to 24.3 g (Table I). During the thaw phase, an increase in the pneumoperitoneum was noted in each case; this was controlled by venting the ports.
es; these may have been situated at the advancing edge of the freezing zone. Many glomeruli were hemorrhagic, and some showed early necrotic changes; fibrin deposition was seen in capillaries and in some interlobular arteries and veins. The hemorrhage extended into the pericaliceal fat. The caliceal, pelvic and ureteric urothelium was intact over the papillae which were not affected by freezing. The urothelium over damaged papillae was almost completely exfoliated; when half of the papilla was damaged, there was corresponding loss of the associated urothelium with sparing of urothelium over intact portions of the papilla. There was focal loss of ureteric epithelium but this was possibly artifactual in origin due to incomplete fixation. Intramural hemorrhage was seen in some large arteries. Adjacent normal renal tissue was clearly demarcated from the hemorrhagic cryoablated areas and the microscopic features were consistent with cell death. Tissue from the control kidney also demonstrated normal renal tubules and glomeruli. The inflammatory response was minimal and consisted of mild focal interstitial polymorphonuclear leukocytes (PMN) infiltration; PMNs were also seen in the endothelium of some blood vessels. Margination of nuclear chromatin resembled the change seen in apoptotic cells. However, the results of the TUNEL technique were negative in these cells.
LIGHT MICROSCOPY Well-defined hemorrhagic zones were present (Fig. 4) in the lower pole of each kidney along with subcapsular hemorrhage. The renal tubules showed early necrotic changes with ill-defined cell borders (Fig. 5) and epithelial exfoliation. Nuclei were vesicular and margination of chromatin was identified in many areas. In some papillae, several terminal collecting ducts showed minimal chang-
ELECTRON MICROSCOPY Ultrastructural examination of kidney from frozen areas showed swelling of tubular epithelial cells. The mitochondria were swollen and cristae destroyed. Many luminal microvilli showed swelling and a number of luminal cells were disrupted. Basal laminae were intact, while the nuclei showed variable degrees of margination of chromatin (Fig.
RESULTS
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FIGURE 3. Hemorrhagic appearance of cryolesion in the lower pole of the kidney. Note clear line of demarcation in the renal parenchyma.
FIGURE 5. Detail of renal tubules showing interstitial hemorrhage, ill-defined borders of cells and margination of nuclear chromatin (arrows) (Hematoxilyn and Eosin 400X).
6). These changes were of such a degree that cell destruction was irreversible. COMMENT The “gold standard” for renal cell carcinoma in patients with a normal contralateral kidney has been radical nephrectomy. Recently, acceptable results have been reported in two large series by major centers utilizing open partial nephrectomy.8,9 Although initially adopted as the procedure of choice in patients with functionally solitary kidneys, the procedure has received acceptance as an option for smaller (,5 cm) polar malignancies. This experience has arisen also in part from the five-fold increase in detection of small (,3 cm) renal tumors between 1974 –1977 and 1982–1985 as a result of more modern cross-sectional imaging modalities.10 Licht and Novick found no signifi164
FIGURE 4. Clearly defined line of demarcation between lesion and intact renal parenchyma (hematoxylin and eosin, 340).
FIGURE 6. Electron micrograph of cryolesion showing pale degenerating cells (D), ill-defined cell borders (arrow) degenerating mitochondria (arrowheads), and marginated nuclear chromatin (M) (X 3,900).
cant differences in survival using this renal-sparing approach, as compared with conventional radical nephrectomy.8 The first laparoscopic nephrectomy was reported by Clayman et al. in 1991.11 Since then, the benefits of shortened hospital stay and convalescence have been well described; yet for most urologists, despite technical innovations, the procedure continues to be more time consuming and technically demanding than its open counterpart. The learning curve is further complicated by the relative rarity of benign nephrectomies. Laparoscopic partial nephrectomy was first reported in 1993 when Winfield et al. performed a laparoscopic partial nephrectomy for a benign, infected kidney.12 While possible, the authors noted that the technical demands and prolonged operative time inherent in this procedure made it somewhat problematic. UROLOGY 51 (Supplement 5A), May 1998
More recently, McDougall et al. reported experience with laparoscopic radical nephrectomy, demonstrating the feasibility of laparoscopic surgery in extirpative renal oncologic surgery.5 Tumors as large as 14 cm have been removed via the laparoscope in an impermeable entrapment sack with low morbidity and without tumor recurrence, albeit at early follow-up.5 In parallel to these strides in laparoscopic urology, the use of percutaneous, ultrasound-guided cryosurgery as a potentially curative procedure for carcinoma of the prostate was reported in 1993.13 Although the data were preliminary, a reproducible result was achievable with ultrasound guidance. Clinical data have continued to accumulate that support cryosurgery as a potential minimally invasive option in locally confined prostate cancer.14 Work has shown that complete ablation of prostatic tissue in dogs is achievable at the microscopic level when using thermosensor control.6 However, reports of severe complications with cryosurgery of the prostate, including urethrorectal fistula have slowed the widespread use of the technique.15 Cryosurgery of the kidney has distinct anatomic advantages over cryosurgery of the prostate. The kidney is surrounded by fat; the kidney may also be freed easily from surrounding structures, unlike the prostate. Similarly, the spherical configuration of many renal tumors follows the geometry of the iceball from a single probe. In 1993, Onik et al. reported the use of ultrasound-guided cryosurgery of the kidney via open laparotomy in the dog.1 This study demonstrated the feasibility of the technique, as well as the ability to monitor the cryolesion with ultrasound. Subsequently, Uchida and colleagues did a descriptive study using percutaneous cryosurgery in dogs and in two human subjects.4 This study showed the increased efficacy of tumor kill in a model of the unperfused kidney due to lack of rapid rewarming from circulating blood. Both patients died from pre-existing metastatic disease. Delworth et al. reported cryosurgery of a midpole renal cell carcinoma in a patient with a solitary kidney via open laparotomy, with success at 1-month follow-up.3 Questions have been raised as to subsequent bleeding with this approach, and the efficacy of tumor kill. Similarly, the ability to maintain adequate surgical margins while controlling damage to surrounding structures remains histologically unproven. This acute study verifies that cell death can be reproducibly and effectively achieved with laparoscopic cryosurgery of the kidney without significant injury to adjacent structures. Overall, the microscopic appearance of these cryolesions demonstrated that the damage was severe and irreversible even though the interval between rewarming of UROLOGY 51 (Supplement 5A), May 1998
tissue and harvesting the kidneys was brief (0 –100 minutes). What remains unclear is the temperature required to achieve adequate cryoablation of tissue and how best to monitor the temperature at the margins of the iceball. In addition, the question arises whether the entire procedure can be carried out percutaneously; Cozzi et al. reported 5 of 10 animals treated with cryoablation had subsequent hemorrhage following thaw.3 Our experience was consistent with this observation. Laparoscopic access to the cryotherapy site may allow for control of minor hemorrhage using avitene pledgets and laparoscopic suturing techniques. Other concerns include damage to adjacent structures, adequacy of tumor staging, delayed hemorrhage, and abscess formation. Further studies are ongoing in this regard. Successful laparoscopic cryosurgery of select polar renal tumors should result in shortened operative times and hospital stays and the ability to perform a curative operation in patients with significant comorbid illnesses as compared with more traditional therapies. The availability of cryosurgical probes for the laparoscope, intraoperative and laparoscopic ultrasound transducers, and sophisticated techniques of selective arterial embolization further increase the potential for clinical application of laparoscopic cryosurgery of the kidney. CONCLUSION This study has evaluated histologically the safety and efficacy of transperitoneal laparoscopic cryoablation of the kidney in the porcine model. Its effectiveness in controlling tumor progression as well as long-term adverse effects, including delayed bleeding and abscess formation, remain untested. We believe, if clinically feasible and effective, laparoscopic cryosurgery of the kidney would provide a simple, minimally invasive technique with which to treat selected renal tumors. ACKNOWLEDGMENTS. We thank Vida Stark for the TUNEL preparation. REFERENCES 1. Onik GM, Reyes G, Cohen JK, and Porterfield B: Ultrasound characteristics of renal cryosurgery. Urology 42: 212– 215, 1993. 2. Cozzi PJ, Lynch WJ, Collins S, Vonthethoff L, and Morris DL. Renal cryotherapy in a sheep model: a feasibilty study. J Urol, 157: 710 –712, 1997. 3. Delworth MG, Pisters LL, Fornage BD, and von Eschenbach, AC: Cryotherapy for renal cell carcinoma and angiomyolipoma. J Urol 155: 252–255, 1996. 4. Uchida M, Imaide Y, Sugimoto K, Uehara H, and Watanabe H: Percutaneous cryosurgery for renal tumors. Br J Urol 75: 132–137, 1995. 5. McDougall EM, Clayman RV, and Elashry OM: Laparoscopic radical nephrectomy for renal tumor: the Washington University experience. J Urol 155: 1180 –1185, 1996. 165
6. Kryger JV, Chinn DO, Messing EM, Siders DB, Lee FT Jr, Zarvan NP, Lee F. Cryosurgical ablation of the prostate in a canine model. Presented at the North Central Section Meeting of the American Urology Association, Minneapolis, 1995. 7. Gavrieli Y, Sherman Y, and Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493–501, 1992. 8. Licht MR, and Novick AC: Nephron sparing surgery for renal cell carcinoma. J Urol 149: 1–7, 1993. 9. Lerner SE, Hawkins CA, Blute ML, Grabner A, Wollan PC, Eickholt JT, and Zincke H: Disease outcome in patients with low stage renal cell carcinoma treated with nephron sparing or radical surgery. J Urol 155: 1868 –1873, 1996. 10. Smith SJ, Bosniak MA, Megibow AJ, Hulnick DH, Horii SC, and Raghavendra BN: Renal cell carcinoma: earlier discovery and increased detection. Radiology 170: 699 –703, 1989.
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11. Clayman RV, Kavoussi LR, Soper NJ, Dierks SM, Meretyk S, Darcy MD, Roemer FD, Pingleton ED, Thomson PG, and Long SR: Laparoscopic nephrectomy: Initial case report. J Urol 146: 278 –282, 1991. 12. Winfield HN, Donovan JF, Godet AS, and Clayman RV: Laparoscopic partial nephrectomy: Initial case report for benign disease. J Endourol 7: 521–526, 1993. 13. Onik GM, Cohen JK, Reyes GD, Rubinsky B, Chang Z, and Baust J: Transrectal ultrasound-guided percutaneous radical cryosurgical ablation of the prostate. Cancer 72: 1291– 1299, 1993. 14. Shinohara K, and Carroll PR: Improved results of cryosurgical ablation of the prostate. J Urol 153: 385A, 1995. 15. Cox RL, and Crawford ED: Complications of cryosurgical ablation of the prostate to treat localized adenocarcinoma of the prostate. Urology 45:932-935, 1996.
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