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Morphologie and functional effects of renal cryoinjury
CRYOBIOLOGY
25, 363-371 (1988)
Morphologic
and Functional Effects of Renal Cryoinjury
GARY W. BARONE AND BRADLEY M. RODGERS’ Division
of Pediatric
Surgery, Children’s Charlottesville,
Medical Center, Virginia 22908
University
of Virginia,
Wilms’ tumor presents with bilateral renal involvement in 10% of patients. With the current treatment protocols for Wilms’ tumor emphasizing maximal preservation of renal parenchyma, cryosurgery may offer an alternative to bilateral tumor resections in these patients. This study was designed to examine the effects of profound cryotherapy on a significant portion of the renal parenchyma, simulating the clinical application of this technique. Eighteen New Zealand white rabbits underwent unilateral nephrectomy, with four serving as controls. The contralateral kidney in the experimental animals was subjected to cryoinjury with a liquid nitrogen probe. Needle thermocouples monitored surface temperature % cm from the margin of the probe tip and renal “core” temperature 1 cm within the renal parenchyma directly beneath the tip. The kidney was cooled until the “core” temperature reached -4O”C, and this was achieved with a probe temperature of - 180°Cfor an average of 11.8 min. All animals tolerated the cryoinjury well and were sacrificed in pairs at 24 and 72 hr, and 1,2,3,4, and 8 weeks. Transient gross hematuria was noted in 25% of the animals and microscopic hematuria in 50%. Serum blood urea nitrogen and creatinine levels reached maximum levels at 72 hr (BUN, 92 f 28 mg%; Creatinine, 7.2 5 1 mg%) and gradually returned toward normal thereafter. The cryolesion at 24 hr resembled a hemorrhagic infarct involving 40% of the kidney. By 8 weeks this lesion had contracted to a fibrotic lesion involving 20% of the kidney by weight. The surrounding uninjured renal parenchyma underwent compensatory hypertrophy. It appears that major cryoinjury is well tolerated by a solitary kidney, and this modality may prove useful in treating a variety of renal lesions including Wilms’ tumors. 0 1988 Academic Press, Inc.
With the introduction of newer and more sophisticated cryosurgical equipment, there has been increasing interest in the wider clinical application of these techniques, particularly in the management of benign and malignant tumors. An important aspect in the management of malignancies is the eradication of neoplastic tissue with preservation of as much of the adjacent normal tissue as possible. Profound cryosurgery, using temperatures below - 40°C has been shown to be effective in treating various superficial malignancies (2). The proper management of Wilms’ tumors has long been of interest to pediatric surgeons. Approximately 10% of these tumors are bi-
lateral at the time of presentation, and treatment of these lesions usually has involved a unilateral radical nephrectomy on the side of the largest tumor with a simultaneous contralateral partial nephrectomy (12). The present study was undertaken to determine if cryotherapy could offer a possible treatment alternative to partial nephrectomy for renal neoplasms especially in a patient with a solitary kidney. METHODS
Eighteen adult white New Zealand rabbits weighing between 3 and 4 kg were anesthetized with intravenous pentobarbital(30 mg/kg) and inhalation halothane. Through a midline laparotomy nine animals underwent a right nephrectomy, and nine underReceived February 1, 1988; accepted March 14, went a left nephrectomy. The weight of the 1988. resected kidney was between 9 and 10 g, i To whom correspondence and reprint requests and there was no significant difference beshould be addressed at Department of Surgery, Box 181, University of Virginia, Charlottesville, VA 22908. tween the weight of the resected right and 363 OOll-2240188$3.00 Copyri&t AU rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
364
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AND
left kidneys. Taking care to protect the renal vessels, the remaining kidney was mobilized to expose its lateral border. Four animals (two right and two left kidneys) served as controls and received no further treatment. In the remaining 14 rabbits cryotherapy was administered with a Frigitronits CE-4 (Frigitronics of Connecticut, Inc., Shelton, CT) instrument utilizing liquid nitrogen as the coolant source. The probe tip consisted of a copper disc 1 cm in diameter. The changes in the temperature in the surrounding renal parenchyma were monitored continuously with two 22-gauge needle thermocouples: one placed on the renal surface 0.5 cm from the edge of the copper disc, and the other placed in the renal “core” 1.0 cm directly beneath the center of the copper disc (Fig. 1). The temperature of the probe tip was measured continuously by means of an internal thermocouple. The cryoinstrument was programmed to achieve a temperature of - 180°C at the copper disc. A site along the lateral border
RODGERS
of the kidney midway between the upper and lower poles was selected for cryotherapy. The probe was applied without pressure to the renal capsule in this region and was activated to cool the renal tissues until the renal “core” temperature reached a cytolethal temperature of - 40°C. Renal blood flow was maintained during the cryotherapy to protect the remainder of the kidney. The probe was then heated with an internal heater to a temperature of +25”C and removed from the renal capsule. During cooling the surrounding tissue was protected from the probe by moist saline sponges. Following cooling the kidney was replaced within the renal fossa, and the abdominal wound was closed. Animals were housed in individual cages and allowed an ad fibitum diet following surgery. They received no antibiotic therapy. Animals were sacrificed in pairs of right and left kidneys at 24 and 72 hr, and 1, 2, 3, 4, and 8 weeks following cryosurgery. The four control animals were sacrificed at 8 weeks following their nephrectomies. Venous blood was drawn for serum blood urea nitrogen (BUN) and creatinine determinations, and urine was collected at the above intervals. At sacrifice the intact cryoinjured kidney and the excised cryolesions were individually weighed, and sections were submitted for microscopic examinations. RESULTS
FIG. 1. Schematic drawing of animal preparation for renal cryoinjury.
All animals tolerated the surgical procedure and cryotherapy without postoperative mortality. Within the first 24 hr gross hematuria was noted in four (23%) of the animals and microscopic hematuria in seven (50%), but all hematuria resolved spontaneously by the end of the first week following cryotherapy. There were no significant differences between the cryoinjured left or right kidneys except for the cooling time, and data from both sides were combined for analysis. All data are expressed as means rt standard errors. Elevations of serum BUN and creatinine
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reached a maximum within 72 hr of cryotherapy (BUN, 92 2 28 mg%; creatinine, 7.2 + 1.0 mg%) and gradually returned toward normal thereafter (Fig. 2). Nonetheless, at 8 weeks following cryoinjury the BUN remained slightly elevated (30 + 2.0 mg%) compared to the g-week control animals (24 * 1.5 mg%) (P < 0.05). The creatinine at 8 weeks following cryotherapy also remained mildly elevated (2.2 + 0.4 mg%) compared with that of the controls (1.6 + 0.2 mg%), but this difference was not statistically significant. The alterations in BUN and creatinine values noted in the animals subjected to cryotherapy closely paralleled the changes in the ratio of the weight of the cryolesion to the weight of the intact kidney (Fig. 2). As this ratio decreased, the BUN and creatinine values also decreased. The mean cooling time to a core temperature of -40°C for all animals was 11.8 ? 2.5 min. The mean cooling time for the right kidney was 9.6 + 1 min and for the left kidney was 13.9 -C 2 min. The difference between right and left was significant (P < 0.05) and may reflect a greater compromise of renal blood flow in mobilizing the right kidney with a longer renal artery. The changes in surface temperatures measured
were quite variable and did not seem to relate to changes in “core” temperature. The rate of cooling, as reflected in “core” temperature, for the right kidney was 7.9YYmin and for the left kidney was 5.5Wmin. The thaw curves had an initial period of rapid warming caused by the probe internal heater, followed by a more gradual temperature increase as the renal blood flow increased to the region of cryoinjury (Fig. 3). Gross sections of the renal lesions revealed a steady maturation of the cryolesion over the 8 weeks (Fig. 4). On Day 1 the cryolesion appeared as a wedge-shaped hemorrhagic infarct involving 40% of the kidney by weight. This lesion progressed over the next 3 to 7 days to a well-defined wedge-shaped infarct accounting for about 55% of the kidney by weight. Over the next 7 weeks there was a steady contracture and fibrosis of this infarcted area, associated with gross hypertrophy of the remaining uninjured renal parenchyma. By 8 weeks the cryoinjured area involved only 20% of the kidney by weight. The weight of the control kidneys at 8 weeks increased 37% when compared to the average weight of the initial nephrectomy kidneys (13.4 f 0.6 vs 9.8 4 0.8 g), and
0
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TIMEIMIN)
FIG. 2. Alterations in renal function and size of cryoinjury over the I-week observation interval.
FIG. 3. Freezing and thawing rate of right and left kidney.
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BARONE AND RODGERS
FIG. 4. Maturation of the gross cryolesion on the lateral border of the kidney over the I-week observation interval.
this signified the expected renal hypertro- compensatory hypertrophy of the noninphy following nephrectomy. In addition, at jured tissue in the cryoinjured kidneys 8 weeks the cryoinjured kidneys weighed when compared to that of the 8-week conabout 20% more than the &week control trol kidney. Changes seen microscopically paralleled kidneys (16.5 vs 13.4 g). After subtracting the weight of the cryoinjured area (20%), the changes noted grossly. Sections of conthe remaining renal parenchyma of the 8- trol and cryotherapy kidney were stained week cryoinjured kidneys weighed the with hematoxylin and eosin, looking for same as the &week control kidneys. Taking changes in the frozen, junctional, and uninto consideration that at 24 hr the cryoin- frozen parenchyma (Figs. 5A-5D). Severe jury involved about 40% of the renal paren- coagulative necrosis of the frozen parenchyma, this indicates a greater degree of chyma was noted to be the only change at
FIG. 5. Microscopic sections at the margin of the cryoinjury at the corticomedullary junction. N, normal kidney. Original magnification, 45X. (A) Twenty-four hours, acute hemorrhage (large arrow) and edema (small arrow) with coagulation necrosis of the tubules. (B) Seventy-two hours, acute inflammatory intihrate (large arrow) with tubular casts (small arrow). (C) Two weeks, proliferation of tibroblasts (asterisk) with necrotic glomeruli and tubules (small arrow). (D) Eight weeks, extensive fibrosis in the cryolesion (asterisk) with deposition of interstitial calcium in the junctional zone (large arrow).
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FIG. Continued.
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369
tery branches. Using vascular cast techniques in human kidneys, Graves (6) has demonstrated the segmental distribution of the major branches of the renal arteries. These vessels are end arteries with virtually no collateral circulation between the segments. The thrombosis of the major vascular supply to the anterior- and posteriormiddle segments in the cryoinjured kidneys would account for the distinctly wedgesloped infarct noted grossly in those areas (Fig. 4). A fourth mechanism which will be discussed later is the possible role of an autoimmune destruction of previously cryoinjured tissue. There are only a few other reports of cryoinjury of the kidney. Bush ef al., in 1964, subjected mobilized kidneys in anesthetized rats to 15 set of submersion in liquid nitrogen (3). The main purpose of this study was to investigate the recovery of rapidly cooled kidneys for renal preservation and future transplantation and not for renal parenchymal destruction. Breining et al., in 1974, described experiments in which the entire kidneys of adult rats were cooled to DISCUSSION - 80°C for 30 set (1). The temperature of The results of these experiments indicate the renal parenchyma was not recorded in that a solitary kidney can tolerate a pro- these experiments, but these authors comfound cryoinjury and recover almost nor- mented on the rapid healing of the renal parenchyma with a barely visible area of mal function. The cryoinjury in a relatively large kidney, such as in a rabbit, appears to fibrosis being evident 2-3 weeks following be caused by several mechanisms. As pre- cryoinjury. Sindelar et al., in 1981, proviously demonstrated in cryoinjured kid- duced a cryolesion in the superior pole of neys (lo), at the cellular level the cooling of the left kidney of adult rats with a blunt tissue to a temperature of - 40°C is charac- 1g-gauge needle and liquid nitrogen (9). The terized microscopically by intracellular wa- kidneys were frozen for 15sec intervals ter crystal formation and intracellular pro- and the authors describe reductions in renal parenchymal temperature to levels of tein coagulation with resulting cellular - 110°C although the exact location of this membrane rupture and cell death. Further cellular destruction is enhanced by local tis- temperature recording was not described. sue anoxia caused by vasoconstriction ini- Histologically, these kidneys demonstrated tially and later by thrombosis of distal arte- the rapid onset of a coagulation necrosis of rioles and venules within the area of cryo- the frozen tissue with parenchymal hemorinjury (9). It appears of even more rhage and the presence of tubal protein importance in the rabbit kidney that lethal casts. At 1 week following cryosurgery few cryoinjury is further caused by the vascular inflammatory cells were noted, and the coocclusion of the larger segmental renal ar- agulated cellular debris was cleared, pre-
24 hr. At 3 to 7 days along with residual hemorrhage, a moderate number of acute inflammatory cells were also present in the area of cryoinjury. For the remaining 7 weeks, there was resolution of the above changes associated with progressive fibrosis and parenchyma mineralization. In the junctional zone between the frozen and the unfrozen parenchyma acute changes occurred for the first 7 days consisting of coagulative necrosis, acute inflammation, and tubular casts. For the remaining 7 weeks there was progressive tubular regeneration and tubular dilation in the remaining viable parenchyma. Interstitial fibrosis in the junctional area was minimal at 1 week, but this progressed to become moderately severe by 8 weeks. A variety of changes were observed in the unfrozen parenchyma. These changes were noted also in the 8week control animals and consisted of multifocal tubular dilation and multifocal tubular regeneration. These changes seem to reflect the renal hypertrophy which occurs after unilateral nephrectomy in the rabbit.
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sumably by the macrophages noted under evidence of hypertrophy which was probably enhanced by the contralateral nephrecthe renal capsule. By 2 weeks following cryoinjury fibrosis of this area was occur- tomy performed in this study. The functional capability of the solitary ring, associated with thickening of the overkidney following cryoinjury is of considerlaying renal capsule. The present study has the advantage of able importance when considering the use producing a more precisely defined cryole- of this modality clinically. Dicker and Shirsion in a solitary kidney and of following ley (4) studied renal function of a solitary functional as well as morphologic changes. kidney following contralateral nephrectoIn this study the renal parenchyma was my in rats. A 30-40% increase in renal cooled until the thermocouple placed 1 cm weight and a similar increase in glomerular beneath the renal capsule measured the cy- filtration rate were noted within approxitotoxic temperature of -40°C. These ex- mately 1 week of nephrectomy. These auperimental conditions mimick more closely thors also noted an enhanced renal hyperthe clinical conditions that would be re- trophy in animals consuming a high protein quired to treat a superficial renal neoplasm. diet. Chemotherapy and radiation therapy Gage has demonstrated that temperatures are important adjuncts in Wilms’ tumor, of -40 to - 50°C are necessary to predict and their influence on renal hypertrophy reliably cellular death (5). The mean time to was evaluated by Walker ef al. (11). These reach cytolethal temperature in these ani- authors noted the normal progression of remals was 11.8 min, and this represents a nal hypertrophy in children receiving checooling rate of approximately 6”C/min. Par- motherapy and radiation following unilattial arterial occlusion may be needed to in- eral nephrectomy for Wilms’ tumor. The crease the rate of freezing and make this findings in our g-week control animals unprocedure more clinically applicable for dergoing a unilateral nephrectomy indicate larger tumors. An internal heater in the approximately a 40% increase in renal probe allowed a relatively rapid thaw and weight and an apparent augmentation of reearly probe disengagement at +25”C. The nal function with serum BUN and creatigross and microscopic changes noted in our nine remaining within the normal range. In kidneys were similar to those reported by addition, in the kidneys subjected to cryoother authors (1, IO). Within 24 hr of cry- injury, the smaller amount of remaining oinjury there was a wedge-shaped hemor- viable renal tissue underwent an even more rhagic injury infarct which involved 40% of substantial morphological and functional the kidney by weight. Histologically the hypertrophy compared with the g-week cryoinjury was characterized by the extra- control kidneys. The weight of the cryoinvasation of red blood cells into the injured jured kidneys also increased about 40% in parenchyma and by the coagulative necro- weight compared to the weight of the basesis of the renal cells in this area. Over the line nephrectomy kidneys, and this exfirst week following cryoinjury this lesion cluded the 20% for the infarcted tissue. actually increased in size to involve 55% of After an initial rise in serum BUN and the renal weight. During the next week, be- creatinine, these parameters steadily approached normal values for the cryoinjured tween 7 and 14 days following cryoinjury, we noted a steady contracture of the in- kidneys, signifying improved renal function. farcted area. By 8 weeks the cryoinjured The issue of enhancement of the tumorarea represented only 20% of the total renal weight and was associated with hypertro- specific immunologic response following phy of the remaining uninjured renal poles cryotherapy has not been addressed by seen grossly and microscopically. Previous these experiments. Several authors have reports of renal cryoinjury did not mention suggested that cryotherapy produces an au-
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toimmune response to the cryoinjured tissues, particularly those of the genitourinary tract (8, 10). Following hepatic and renal cryotherapy, Helpap et al. have demonstrated a marked immunologic proliferation within the spleen (8). They suggested two possible mechanisms for how cryotherapy may initiate a primary immunologic response. Cryoinjury leads to cellular membrane rupture with the release of intracellular, sequestered contents. These fractions may not have had contact previously with the immune system and may act as autoantigens. The other possible mechanism is that cooling modifies cellular antigens by the denaturation of protein and polypeptides. The immune system then recognizes these as foreign proteins and initiates an autoantigen-antibody reaction. Some clinical studies also have suggested the possibility of a tumor-specific immune response following cryotherapy (7). Although more studies will be necessary to clarify this important area, this remains a potential benefit of the use of cryotherapy for the treatment of renal neoplasms. The results of these studies indicate that a solitary kidney is able to withstand a significant cryoinjury, ultimately involving slightly more than half of the kidney by weight. Nearly complete morphologic and functional recovery occurs in these kidneys over an g-week interval following cryoinjury with significant compensatory hypertrophy of the unfrozen tissue. These results strongly suggest that cryotherapy may be useful in the treatment of renal neoplasms. Clinically, cryotherapy has the potential of eradicating localized neoplastic lesions while preserving surrounding normal functioning parenchyma. This may be particularly applicable in the management of multifocal renal lesions. Also, cryotherapy could be used for primary debulking of renal tumors, possibly followed by chemotherapy as for Wilms’ tumors or as an adjunct for destroying residual tumor after surgical debulking. With increasing poten-
tial for immunotherapy in the management of neoplastic disease, the enhanced immune response elicited by cryotherapy may be an added benefit in the management of renal neoplasms. The application of cryotherapy to an experimental renal tumor model will help to define better the potential utility of this modality. REFERENCES
1. Breining, H., Helpap, B., Minderjahan, A., and Lymberopoulos, S. Histological and autoradiographic findings in cryonecrosis of the liver and kidney. Cryobiology 11, 519-525 (1974). 2. Bullock, J. D., Beard, C., and Sullivan, J. H. Cryotherapy of basal cell carcinoma in oculoplastic surgery. Amer. J. Ophthalmol. 82, 848850 (1976). 3. Bush, I. M., Santoni, E., Lieberman, P. H., Cahan, W. Cl., and Whitmore, W. F. Some effects of freezing the rat kidney in situ. Cryobiology 2, 163-170 (1964). 4. Dicker, S., and Shirley, D. Mechanism of compensatory renal hypertrophy. J. Physiol. 219, 507-523 (1971). 5. Gage, A. Cryosurgery of oral and pharyngeal carcinoma. Amer. J. Surg. 118, 669-672 (1969). 6. Graves, F. T. The anatomy of the intrarenal arteries and its application to segmental resection of the kidney. Brit. J. Surg. 42, 132-139 (1954). 7. Helpap, B., Groules, V., Lange, O., Breining, H., and Lymberopoulos, S. Morphologic and cell kinetic investigations of the spleen after repeated in situ freezing of liver and kidney. Pathol. Res. Pratt. 164, 167-177 (1979). 8. Helpap, B., Groules, V., Yamashita, K., and Breining, H. The proliferative response of the spleen in cryosurgery. Cryobiology 13, 54-60 (1976). 9. Kreyberg, L. Stasis and necrosis. Stand. J. Clin. Lab. Invest. lS(Supp1. 71), l-26 (1963). 10. Sindelar, W., Javadpour, N., and Bagley, D. Histological and ultrastructural changes in rat kidney after cryosurgery. J. Surg. Oncol. 18, 363379 (1981). 11. Walker, R. D., Talbert, J. L., Rodgers, B. M., Reid, F., and Richard, G. A. Compensatory renal growth and function in post-nephrectomized Wilms’ tumor patients. J. Ural. 19, 127-130 (1982). 12. Wasiljeir, B., Besser, A., and Raffensperger, J. Treatment of bilateral Wilms’ tumor-A 22 year experience. J. Pediatr. Surg. 17, 265-268 (1982).
25, 363-371 (1988)
Morphologic
and Functional Effects of Renal Cryoinjury
GARY W. BARONE AND BRADLEY M. RODGERS’ Division
of Pediatric
Surgery, Children’s Charlottesville,
Medical Center, Virginia 22908
University
of Virginia,
Wilms’ tumor presents with bilateral renal involvement in 10% of patients. With the current treatment protocols for Wilms’ tumor emphasizing maximal preservation of renal parenchyma, cryosurgery may offer an alternative to bilateral tumor resections in these patients. This study was designed to examine the effects of profound cryotherapy on a significant portion of the renal parenchyma, simulating the clinical application of this technique. Eighteen New Zealand white rabbits underwent unilateral nephrectomy, with four serving as controls. The contralateral kidney in the experimental animals was subjected to cryoinjury with a liquid nitrogen probe. Needle thermocouples monitored surface temperature % cm from the margin of the probe tip and renal “core” temperature 1 cm within the renal parenchyma directly beneath the tip. The kidney was cooled until the “core” temperature reached -4O”C, and this was achieved with a probe temperature of - 180°Cfor an average of 11.8 min. All animals tolerated the cryoinjury well and were sacrificed in pairs at 24 and 72 hr, and 1,2,3,4, and 8 weeks. Transient gross hematuria was noted in 25% of the animals and microscopic hematuria in 50%. Serum blood urea nitrogen and creatinine levels reached maximum levels at 72 hr (BUN, 92 f 28 mg%; Creatinine, 7.2 5 1 mg%) and gradually returned toward normal thereafter. The cryolesion at 24 hr resembled a hemorrhagic infarct involving 40% of the kidney. By 8 weeks this lesion had contracted to a fibrotic lesion involving 20% of the kidney by weight. The surrounding uninjured renal parenchyma underwent compensatory hypertrophy. It appears that major cryoinjury is well tolerated by a solitary kidney, and this modality may prove useful in treating a variety of renal lesions including Wilms’ tumors. 0 1988 Academic Press, Inc.
With the introduction of newer and more sophisticated cryosurgical equipment, there has been increasing interest in the wider clinical application of these techniques, particularly in the management of benign and malignant tumors. An important aspect in the management of malignancies is the eradication of neoplastic tissue with preservation of as much of the adjacent normal tissue as possible. Profound cryosurgery, using temperatures below - 40°C has been shown to be effective in treating various superficial malignancies (2). The proper management of Wilms’ tumors has long been of interest to pediatric surgeons. Approximately 10% of these tumors are bi-
lateral at the time of presentation, and treatment of these lesions usually has involved a unilateral radical nephrectomy on the side of the largest tumor with a simultaneous contralateral partial nephrectomy (12). The present study was undertaken to determine if cryotherapy could offer a possible treatment alternative to partial nephrectomy for renal neoplasms especially in a patient with a solitary kidney. METHODS
Eighteen adult white New Zealand rabbits weighing between 3 and 4 kg were anesthetized with intravenous pentobarbital(30 mg/kg) and inhalation halothane. Through a midline laparotomy nine animals underwent a right nephrectomy, and nine underReceived February 1, 1988; accepted March 14, went a left nephrectomy. The weight of the 1988. resected kidney was between 9 and 10 g, i To whom correspondence and reprint requests and there was no significant difference beshould be addressed at Department of Surgery, Box 181, University of Virginia, Charlottesville, VA 22908. tween the weight of the resected right and 363 OOll-2240188$3.00 Copyri&t AU rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
364
BARONE
AND
left kidneys. Taking care to protect the renal vessels, the remaining kidney was mobilized to expose its lateral border. Four animals (two right and two left kidneys) served as controls and received no further treatment. In the remaining 14 rabbits cryotherapy was administered with a Frigitronits CE-4 (Frigitronics of Connecticut, Inc., Shelton, CT) instrument utilizing liquid nitrogen as the coolant source. The probe tip consisted of a copper disc 1 cm in diameter. The changes in the temperature in the surrounding renal parenchyma were monitored continuously with two 22-gauge needle thermocouples: one placed on the renal surface 0.5 cm from the edge of the copper disc, and the other placed in the renal “core” 1.0 cm directly beneath the center of the copper disc (Fig. 1). The temperature of the probe tip was measured continuously by means of an internal thermocouple. The cryoinstrument was programmed to achieve a temperature of - 180°C at the copper disc. A site along the lateral border
RODGERS
of the kidney midway between the upper and lower poles was selected for cryotherapy. The probe was applied without pressure to the renal capsule in this region and was activated to cool the renal tissues until the renal “core” temperature reached a cytolethal temperature of - 40°C. Renal blood flow was maintained during the cryotherapy to protect the remainder of the kidney. The probe was then heated with an internal heater to a temperature of +25”C and removed from the renal capsule. During cooling the surrounding tissue was protected from the probe by moist saline sponges. Following cooling the kidney was replaced within the renal fossa, and the abdominal wound was closed. Animals were housed in individual cages and allowed an ad fibitum diet following surgery. They received no antibiotic therapy. Animals were sacrificed in pairs of right and left kidneys at 24 and 72 hr, and 1, 2, 3, 4, and 8 weeks following cryosurgery. The four control animals were sacrificed at 8 weeks following their nephrectomies. Venous blood was drawn for serum blood urea nitrogen (BUN) and creatinine determinations, and urine was collected at the above intervals. At sacrifice the intact cryoinjured kidney and the excised cryolesions were individually weighed, and sections were submitted for microscopic examinations. RESULTS
FIG. 1. Schematic drawing of animal preparation for renal cryoinjury.
All animals tolerated the surgical procedure and cryotherapy without postoperative mortality. Within the first 24 hr gross hematuria was noted in four (23%) of the animals and microscopic hematuria in seven (50%), but all hematuria resolved spontaneously by the end of the first week following cryotherapy. There were no significant differences between the cryoinjured left or right kidneys except for the cooling time, and data from both sides were combined for analysis. All data are expressed as means rt standard errors. Elevations of serum BUN and creatinine
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reached a maximum within 72 hr of cryotherapy (BUN, 92 2 28 mg%; creatinine, 7.2 + 1.0 mg%) and gradually returned toward normal thereafter (Fig. 2). Nonetheless, at 8 weeks following cryoinjury the BUN remained slightly elevated (30 + 2.0 mg%) compared to the g-week control animals (24 * 1.5 mg%) (P < 0.05). The creatinine at 8 weeks following cryotherapy also remained mildly elevated (2.2 + 0.4 mg%) compared with that of the controls (1.6 + 0.2 mg%), but this difference was not statistically significant. The alterations in BUN and creatinine values noted in the animals subjected to cryotherapy closely paralleled the changes in the ratio of the weight of the cryolesion to the weight of the intact kidney (Fig. 2). As this ratio decreased, the BUN and creatinine values also decreased. The mean cooling time to a core temperature of -40°C for all animals was 11.8 ? 2.5 min. The mean cooling time for the right kidney was 9.6 + 1 min and for the left kidney was 13.9 -C 2 min. The difference between right and left was significant (P < 0.05) and may reflect a greater compromise of renal blood flow in mobilizing the right kidney with a longer renal artery. The changes in surface temperatures measured
were quite variable and did not seem to relate to changes in “core” temperature. The rate of cooling, as reflected in “core” temperature, for the right kidney was 7.9YYmin and for the left kidney was 5.5Wmin. The thaw curves had an initial period of rapid warming caused by the probe internal heater, followed by a more gradual temperature increase as the renal blood flow increased to the region of cryoinjury (Fig. 3). Gross sections of the renal lesions revealed a steady maturation of the cryolesion over the 8 weeks (Fig. 4). On Day 1 the cryolesion appeared as a wedge-shaped hemorrhagic infarct involving 40% of the kidney by weight. This lesion progressed over the next 3 to 7 days to a well-defined wedge-shaped infarct accounting for about 55% of the kidney by weight. Over the next 7 weeks there was a steady contracture and fibrosis of this infarcted area, associated with gross hypertrophy of the remaining uninjured renal parenchyma. By 8 weeks the cryoinjured area involved only 20% of the kidney by weight. The weight of the control kidneys at 8 weeks increased 37% when compared to the average weight of the initial nephrectomy kidneys (13.4 f 0.6 vs 9.8 4 0.8 g), and
0
5
IO
I5
20
25
30
TIMEIMIN)
FIG. 2. Alterations in renal function and size of cryoinjury over the I-week observation interval.
FIG. 3. Freezing and thawing rate of right and left kidney.
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FIG. 4. Maturation of the gross cryolesion on the lateral border of the kidney over the I-week observation interval.
this signified the expected renal hypertro- compensatory hypertrophy of the noninphy following nephrectomy. In addition, at jured tissue in the cryoinjured kidneys 8 weeks the cryoinjured kidneys weighed when compared to that of the 8-week conabout 20% more than the &week control trol kidney. Changes seen microscopically paralleled kidneys (16.5 vs 13.4 g). After subtracting the weight of the cryoinjured area (20%), the changes noted grossly. Sections of conthe remaining renal parenchyma of the 8- trol and cryotherapy kidney were stained week cryoinjured kidneys weighed the with hematoxylin and eosin, looking for same as the &week control kidneys. Taking changes in the frozen, junctional, and uninto consideration that at 24 hr the cryoin- frozen parenchyma (Figs. 5A-5D). Severe jury involved about 40% of the renal paren- coagulative necrosis of the frozen parenchyma, this indicates a greater degree of chyma was noted to be the only change at
FIG. 5. Microscopic sections at the margin of the cryoinjury at the corticomedullary junction. N, normal kidney. Original magnification, 45X. (A) Twenty-four hours, acute hemorrhage (large arrow) and edema (small arrow) with coagulation necrosis of the tubules. (B) Seventy-two hours, acute inflammatory intihrate (large arrow) with tubular casts (small arrow). (C) Two weeks, proliferation of tibroblasts (asterisk) with necrotic glomeruli and tubules (small arrow). (D) Eight weeks, extensive fibrosis in the cryolesion (asterisk) with deposition of interstitial calcium in the junctional zone (large arrow).
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FIG. Continued.
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tery branches. Using vascular cast techniques in human kidneys, Graves (6) has demonstrated the segmental distribution of the major branches of the renal arteries. These vessels are end arteries with virtually no collateral circulation between the segments. The thrombosis of the major vascular supply to the anterior- and posteriormiddle segments in the cryoinjured kidneys would account for the distinctly wedgesloped infarct noted grossly in those areas (Fig. 4). A fourth mechanism which will be discussed later is the possible role of an autoimmune destruction of previously cryoinjured tissue. There are only a few other reports of cryoinjury of the kidney. Bush ef al., in 1964, subjected mobilized kidneys in anesthetized rats to 15 set of submersion in liquid nitrogen (3). The main purpose of this study was to investigate the recovery of rapidly cooled kidneys for renal preservation and future transplantation and not for renal parenchymal destruction. Breining et al., in 1974, described experiments in which the entire kidneys of adult rats were cooled to DISCUSSION - 80°C for 30 set (1). The temperature of The results of these experiments indicate the renal parenchyma was not recorded in that a solitary kidney can tolerate a pro- these experiments, but these authors comfound cryoinjury and recover almost nor- mented on the rapid healing of the renal parenchyma with a barely visible area of mal function. The cryoinjury in a relatively large kidney, such as in a rabbit, appears to fibrosis being evident 2-3 weeks following be caused by several mechanisms. As pre- cryoinjury. Sindelar et al., in 1981, proviously demonstrated in cryoinjured kid- duced a cryolesion in the superior pole of neys (lo), at the cellular level the cooling of the left kidney of adult rats with a blunt tissue to a temperature of - 40°C is charac- 1g-gauge needle and liquid nitrogen (9). The terized microscopically by intracellular wa- kidneys were frozen for 15sec intervals ter crystal formation and intracellular pro- and the authors describe reductions in renal parenchymal temperature to levels of tein coagulation with resulting cellular - 110°C although the exact location of this membrane rupture and cell death. Further cellular destruction is enhanced by local tis- temperature recording was not described. sue anoxia caused by vasoconstriction ini- Histologically, these kidneys demonstrated tially and later by thrombosis of distal arte- the rapid onset of a coagulation necrosis of rioles and venules within the area of cryo- the frozen tissue with parenchymal hemorinjury (9). It appears of even more rhage and the presence of tubal protein importance in the rabbit kidney that lethal casts. At 1 week following cryosurgery few cryoinjury is further caused by the vascular inflammatory cells were noted, and the coocclusion of the larger segmental renal ar- agulated cellular debris was cleared, pre-
24 hr. At 3 to 7 days along with residual hemorrhage, a moderate number of acute inflammatory cells were also present in the area of cryoinjury. For the remaining 7 weeks, there was resolution of the above changes associated with progressive fibrosis and parenchyma mineralization. In the junctional zone between the frozen and the unfrozen parenchyma acute changes occurred for the first 7 days consisting of coagulative necrosis, acute inflammation, and tubular casts. For the remaining 7 weeks there was progressive tubular regeneration and tubular dilation in the remaining viable parenchyma. Interstitial fibrosis in the junctional area was minimal at 1 week, but this progressed to become moderately severe by 8 weeks. A variety of changes were observed in the unfrozen parenchyma. These changes were noted also in the 8week control animals and consisted of multifocal tubular dilation and multifocal tubular regeneration. These changes seem to reflect the renal hypertrophy which occurs after unilateral nephrectomy in the rabbit.
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sumably by the macrophages noted under evidence of hypertrophy which was probably enhanced by the contralateral nephrecthe renal capsule. By 2 weeks following cryoinjury fibrosis of this area was occur- tomy performed in this study. The functional capability of the solitary ring, associated with thickening of the overkidney following cryoinjury is of considerlaying renal capsule. The present study has the advantage of able importance when considering the use producing a more precisely defined cryole- of this modality clinically. Dicker and Shirsion in a solitary kidney and of following ley (4) studied renal function of a solitary functional as well as morphologic changes. kidney following contralateral nephrectoIn this study the renal parenchyma was my in rats. A 30-40% increase in renal cooled until the thermocouple placed 1 cm weight and a similar increase in glomerular beneath the renal capsule measured the cy- filtration rate were noted within approxitotoxic temperature of -40°C. These ex- mately 1 week of nephrectomy. These auperimental conditions mimick more closely thors also noted an enhanced renal hyperthe clinical conditions that would be re- trophy in animals consuming a high protein quired to treat a superficial renal neoplasm. diet. Chemotherapy and radiation therapy Gage has demonstrated that temperatures are important adjuncts in Wilms’ tumor, of -40 to - 50°C are necessary to predict and their influence on renal hypertrophy reliably cellular death (5). The mean time to was evaluated by Walker ef al. (11). These reach cytolethal temperature in these ani- authors noted the normal progression of remals was 11.8 min, and this represents a nal hypertrophy in children receiving checooling rate of approximately 6”C/min. Par- motherapy and radiation following unilattial arterial occlusion may be needed to in- eral nephrectomy for Wilms’ tumor. The crease the rate of freezing and make this findings in our g-week control animals unprocedure more clinically applicable for dergoing a unilateral nephrectomy indicate larger tumors. An internal heater in the approximately a 40% increase in renal probe allowed a relatively rapid thaw and weight and an apparent augmentation of reearly probe disengagement at +25”C. The nal function with serum BUN and creatigross and microscopic changes noted in our nine remaining within the normal range. In kidneys were similar to those reported by addition, in the kidneys subjected to cryoother authors (1, IO). Within 24 hr of cry- injury, the smaller amount of remaining oinjury there was a wedge-shaped hemor- viable renal tissue underwent an even more rhagic injury infarct which involved 40% of substantial morphological and functional the kidney by weight. Histologically the hypertrophy compared with the g-week cryoinjury was characterized by the extra- control kidneys. The weight of the cryoinvasation of red blood cells into the injured jured kidneys also increased about 40% in parenchyma and by the coagulative necro- weight compared to the weight of the basesis of the renal cells in this area. Over the line nephrectomy kidneys, and this exfirst week following cryoinjury this lesion cluded the 20% for the infarcted tissue. actually increased in size to involve 55% of After an initial rise in serum BUN and the renal weight. During the next week, be- creatinine, these parameters steadily approached normal values for the cryoinjured tween 7 and 14 days following cryoinjury, we noted a steady contracture of the in- kidneys, signifying improved renal function. farcted area. By 8 weeks the cryoinjured The issue of enhancement of the tumorarea represented only 20% of the total renal weight and was associated with hypertro- specific immunologic response following phy of the remaining uninjured renal poles cryotherapy has not been addressed by seen grossly and microscopically. Previous these experiments. Several authors have reports of renal cryoinjury did not mention suggested that cryotherapy produces an au-
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toimmune response to the cryoinjured tissues, particularly those of the genitourinary tract (8, 10). Following hepatic and renal cryotherapy, Helpap et al. have demonstrated a marked immunologic proliferation within the spleen (8). They suggested two possible mechanisms for how cryotherapy may initiate a primary immunologic response. Cryoinjury leads to cellular membrane rupture with the release of intracellular, sequestered contents. These fractions may not have had contact previously with the immune system and may act as autoantigens. The other possible mechanism is that cooling modifies cellular antigens by the denaturation of protein and polypeptides. The immune system then recognizes these as foreign proteins and initiates an autoantigen-antibody reaction. Some clinical studies also have suggested the possibility of a tumor-specific immune response following cryotherapy (7). Although more studies will be necessary to clarify this important area, this remains a potential benefit of the use of cryotherapy for the treatment of renal neoplasms. The results of these studies indicate that a solitary kidney is able to withstand a significant cryoinjury, ultimately involving slightly more than half of the kidney by weight. Nearly complete morphologic and functional recovery occurs in these kidneys over an g-week interval following cryoinjury with significant compensatory hypertrophy of the unfrozen tissue. These results strongly suggest that cryotherapy may be useful in the treatment of renal neoplasms. Clinically, cryotherapy has the potential of eradicating localized neoplastic lesions while preserving surrounding normal functioning parenchyma. This may be particularly applicable in the management of multifocal renal lesions. Also, cryotherapy could be used for primary debulking of renal tumors, possibly followed by chemotherapy as for Wilms’ tumors or as an adjunct for destroying residual tumor after surgical debulking. With increasing poten-
tial for immunotherapy in the management of neoplastic disease, the enhanced immune response elicited by cryotherapy may be an added benefit in the management of renal neoplasms. The application of cryotherapy to an experimental renal tumor model will help to define better the potential utility of this modality. REFERENCES
1. Breining, H., Helpap, B., Minderjahan, A., and Lymberopoulos, S. Histological and autoradiographic findings in cryonecrosis of the liver and kidney. Cryobiology 11, 519-525 (1974). 2. Bullock, J. D., Beard, C., and Sullivan, J. H. Cryotherapy of basal cell carcinoma in oculoplastic surgery. Amer. J. Ophthalmol. 82, 848850 (1976). 3. Bush, I. M., Santoni, E., Lieberman, P. H., Cahan, W. Cl., and Whitmore, W. F. Some effects of freezing the rat kidney in situ. Cryobiology 2, 163-170 (1964). 4. Dicker, S., and Shirley, D. Mechanism of compensatory renal hypertrophy. J. Physiol. 219, 507-523 (1971). 5. Gage, A. Cryosurgery of oral and pharyngeal carcinoma. Amer. J. Surg. 118, 669-672 (1969). 6. Graves, F. T. The anatomy of the intrarenal arteries and its application to segmental resection of the kidney. Brit. J. Surg. 42, 132-139 (1954). 7. Helpap, B., Groules, V., Lange, O., Breining, H., and Lymberopoulos, S. Morphologic and cell kinetic investigations of the spleen after repeated in situ freezing of liver and kidney. Pathol. Res. Pratt. 164, 167-177 (1979). 8. Helpap, B., Groules, V., Yamashita, K., and Breining, H. The proliferative response of the spleen in cryosurgery. Cryobiology 13, 54-60 (1976). 9. Kreyberg, L. Stasis and necrosis. Stand. J. Clin. Lab. Invest. lS(Supp1. 71), l-26 (1963). 10. Sindelar, W., Javadpour, N., and Bagley, D. Histological and ultrastructural changes in rat kidney after cryosurgery. J. Surg. Oncol. 18, 363379 (1981). 11. Walker, R. D., Talbert, J. L., Rodgers, B. M., Reid, F., and Richard, G. A. Compensatory renal growth and function in post-nephrectomized Wilms’ tumor patients. J. Ural. 19, 127-130 (1982). 12. Wasiljeir, B., Besser, A., and Raffensperger, J. Treatment of bilateral Wilms’ tumor-A 22 year experience. J. Pediatr. Surg. 17, 265-268 (1982).