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Ultrasonic characterization of a cryosurgical process in a gelatin medium
ABSTRACTS,
2lst ANNUAL
Cooling and warming rates affect the viability of cryopreserved organs and tissues, and are therefore of significant interest to medical cryobiologists. Because tests of skin viability relying on graft “take” may yield falsely positive results, we have undertaken a comparison of the effect of different warming rates on the metabolic viability of cryopreserved skin as measured by the conversion of radiolabeled glucose to radiolabeled carbon dioxide (S. R. May and F. A. DeClement. Cryobiology 19, 362-371. 1982). Viable skin in cryopreservation medium composed of Eagle’s Minimal Essential Medium with IO% human pooled sera. antibiotics. and 15% glycerol. was placed in flat packets and frozen at a controlled rate of - 1°C min ’ down to - 100°C. transferred to liquid nitrogen (~ 196”C), then warmed by packet immersion with propeller mixing in regulated constant water temperatures of 3. 4, 5. 10, 15. 20, 25. 30. 35. 37, 40. 42. 4.5. 50. 55. 60, and 65°C. Skin warming rate5 (w) in “C min-’ varied from 30°C to 882°C min- ’ and could be expressed as a function of the constant warming temperature (n according to the formula: W = 12.62T. The maximum skin viability (76-7X% of normal fresh skin) was preserved at warming temperatures of IO’C (rate = 95°C min-‘) to 20°C (rate = 26O”C min-I). Skin viability decreased rapidly at warming temperatures of 3-K (rate = 30-53°C min-‘), where it was only 52-70s of normal. Skin viability also decreased rapidly when warming temperatures above 37°C were employed. A warming temperature of 40°C (rate = 501°C min-‘) decreased viability to 47% of normal, 42°C (rate = 514°C min-‘) decreased viability to 38% of normal, and 45°C (rate = 558°C min-‘) decreased viability to 21% of normal. At warming temperatures of 60-6X (rate = 758-882°C min-‘1. no viability was preserved. The optimum warming rates found in these experiments ( +Y5 to + 260°C min-‘) are higher than those established by Billingham and Medawar in 1952 (+80 to + 100°C min-‘1. and by Lehr er N/. in 1964 (+50 to +7O”C min-I). XX. Reccmdimtion of the And und the Esophged Stenosis from Unresectuhle Conc,er: A Report c?fTwo Cases. S. SUMIDA. T. TAKIHAKA, F. MORISHIGE,* AND T. NAKAMURA* (Division of
Cardiovascular Surgery, National Central Hospital, and *Nakamura Hospital, Fukuoka. Japan
Fukuoka Memorial
A 7%year-old female with unresectable adenocarcinema of the anal canal after colostomy was treated by cryosurgery using liquid nitrogen. A cryoprobe was inserted into the narrowed canal after dilation. A triple freeze-thaw cycle was employed and resulted in cryonecrosis. In 2 weeks, the cancerous stenosis was necrotized and recanalized. A new epithelialized canal was formed in 2 months. The patient was discharged
MEETING
715
in 3 months after cryosurgery. Another case involved a 64-year-old male with unresectable squamous cell cancer of the intrathoracic midesophagus with pulmonary metastasis. Under general anesthesia, a liquidnitrogen cryoprobe was inserted through the mouth into the central portion of the cancer and frozen three times using an intrathoracic maneuver and the aid of an assistant. Cryonecrosis resulted in recanalization in a week. The patient was discharged a month after cryosurgery and his dysphagia disappeared. These cases involved unresectable cancers and demonstrated that cryosurgery could be applied for palliation. 89. Replrrcernent of Blood Vessels: Arterial Grgfi.fbl/owing Cyopreserl’ation in Vrrsc~ulur Acc,es.c Surgc~,.y. EHRSAM.+ GONAKI.’
PH. LEBALID,* L. ODDOU.* A. A. LARbsE.t L. FKAPPAK~.* J. M.w A. GEL.F,T.$ AND J. M. DUBERNARD:
(“Laboratoire de Microchirurgie INSERM U 80. H6pital Edouard Herriot. 69374 Lyon. Cedex. 08 France: -i-Unite de Cryobiologie, Centre de Transfusion Sanguine de Lyon. France: and *Service d’Urologie et de Chirurgie de la Transplantation. Hbpital Edouard Herriot. 69374 Lyon, Cedex 08. France). Twenty rat aortas were aseptically removed and preserved by freezing in liquid phase: The grafts were placed in a cryoprotective solution (DMSO) at varying concentrations and were subsequently frozen in a programmed fashion (temperature lowered at a rate of 1”Cimin and Kimin thereafter until a temperature of - 180°C was achieved). The frozen aortas were preserved in liquid nitrogen until used. These were thawed by simple heating to 37°C. A small segment was removed for histological observation by optical and electron microscopy and the remaining portion of the graft was implanted in rats of the same strain (IO isografts) or of different strains (IO allografts). The preserved grafts showed excellent conservation of the histological structure of the arterial walls (isolated endothelial lesions). The isografted vessels were all permeable after 20 days and 60% of these exhibited excellent histological aspects with respect to the allografts, the majority of the grafts showed important phasmocyte infiltration. These results suggest that this method of preservation is superior to those currently used for the conservation of blood vessels. Another long-term series of five isografted animals sacrificed between 90 to 255 days following the graft confirms the excellent evolution of the arterial grafts. This method of arterial cryopreservation is currently used for the realization of seven human arteriovenous fistulae for hemodialysis by using preserved femoral arteries obtained from cadavers.
716
ABSTRACTS,
2lst ANNUAL
G. M. ONIK,t w. K. HODDlCK,t AND BINSKY* (*Department of Mechanical
B. RUEngineering, University of California, Berkeley, California 94720and tDepartment of Radiology. University of California, San Francisco. California 94143). The use of low temperatures to destroy abnormal tissue (e.g., malignant tumors) is the basis for cryosurgical procedures. Cryosurgery would have the potential for treating some unresectable tumors in the liver, prostate, or brain were it not for the lack of a reliable means to monitor precisely the extent of the frozen region in real time. The use of ultrasound imaging technology has been proposed for this purpose.
MEETING
Experiments on transparent bovine gelatin samples were conducted to determine feasibility and accuracy of the method. A cryosurgical probe with a closed cylindrical tip was used to produce a cryolesion at one face of a rectangular solid gelatin sample. An ultrasound transducer was located at the opposite face to monitor the freezing process. A hemispherical frozen region in the gelatin was clearly defined in the ultrasonogram as a semicircular hyperechoic rim. The location of the change of phase interface is at the inner margin of this rim. Objects such as thermocouples placed in the gelatin prior to freezing, were visible in the ultrasonic image of the frozen region. Both 3.5 and 5 MHz ultrasound transducers were used. Size measurements of the frozen region correlated with the ultrasound image to within 0.7 mm.
2lst ANNUAL
Cooling and warming rates affect the viability of cryopreserved organs and tissues, and are therefore of significant interest to medical cryobiologists. Because tests of skin viability relying on graft “take” may yield falsely positive results, we have undertaken a comparison of the effect of different warming rates on the metabolic viability of cryopreserved skin as measured by the conversion of radiolabeled glucose to radiolabeled carbon dioxide (S. R. May and F. A. DeClement. Cryobiology 19, 362-371. 1982). Viable skin in cryopreservation medium composed of Eagle’s Minimal Essential Medium with IO% human pooled sera. antibiotics. and 15% glycerol. was placed in flat packets and frozen at a controlled rate of - 1°C min ’ down to - 100°C. transferred to liquid nitrogen (~ 196”C), then warmed by packet immersion with propeller mixing in regulated constant water temperatures of 3. 4, 5. 10, 15. 20, 25. 30. 35. 37, 40. 42. 4.5. 50. 55. 60, and 65°C. Skin warming rate5 (w) in “C min-’ varied from 30°C to 882°C min- ’ and could be expressed as a function of the constant warming temperature (n according to the formula: W = 12.62T. The maximum skin viability (76-7X% of normal fresh skin) was preserved at warming temperatures of IO’C (rate = 95°C min-‘) to 20°C (rate = 26O”C min-I). Skin viability decreased rapidly at warming temperatures of 3-K (rate = 30-53°C min-‘), where it was only 52-70s of normal. Skin viability also decreased rapidly when warming temperatures above 37°C were employed. A warming temperature of 40°C (rate = 501°C min-‘) decreased viability to 47% of normal, 42°C (rate = 514°C min-‘) decreased viability to 38% of normal, and 45°C (rate = 558°C min-‘) decreased viability to 21% of normal. At warming temperatures of 60-6X (rate = 758-882°C min-‘1. no viability was preserved. The optimum warming rates found in these experiments ( +Y5 to + 260°C min-‘) are higher than those established by Billingham and Medawar in 1952 (+80 to + 100°C min-‘1. and by Lehr er N/. in 1964 (+50 to +7O”C min-I). XX. Reccmdimtion of the And und the Esophged Stenosis from Unresectuhle Conc,er: A Report c?fTwo Cases. S. SUMIDA. T. TAKIHAKA, F. MORISHIGE,* AND T. NAKAMURA* (Division of
Cardiovascular Surgery, National Central Hospital, and *Nakamura Hospital, Fukuoka. Japan
Fukuoka Memorial
A 7%year-old female with unresectable adenocarcinema of the anal canal after colostomy was treated by cryosurgery using liquid nitrogen. A cryoprobe was inserted into the narrowed canal after dilation. A triple freeze-thaw cycle was employed and resulted in cryonecrosis. In 2 weeks, the cancerous stenosis was necrotized and recanalized. A new epithelialized canal was formed in 2 months. The patient was discharged
MEETING
715
in 3 months after cryosurgery. Another case involved a 64-year-old male with unresectable squamous cell cancer of the intrathoracic midesophagus with pulmonary metastasis. Under general anesthesia, a liquidnitrogen cryoprobe was inserted through the mouth into the central portion of the cancer and frozen three times using an intrathoracic maneuver and the aid of an assistant. Cryonecrosis resulted in recanalization in a week. The patient was discharged a month after cryosurgery and his dysphagia disappeared. These cases involved unresectable cancers and demonstrated that cryosurgery could be applied for palliation. 89. Replrrcernent of Blood Vessels: Arterial Grgfi.fbl/owing Cyopreserl’ation in Vrrsc~ulur Acc,es.c Surgc~,.y. EHRSAM.+ GONAKI.’
PH. LEBALID,* L. ODDOU.* A. A. LARbsE.t L. FKAPPAK~.* J. M.w A. GEL.F,T.$ AND J. M. DUBERNARD:
(“Laboratoire de Microchirurgie INSERM U 80. H6pital Edouard Herriot. 69374 Lyon. Cedex. 08 France: -i-Unite de Cryobiologie, Centre de Transfusion Sanguine de Lyon. France: and *Service d’Urologie et de Chirurgie de la Transplantation. Hbpital Edouard Herriot. 69374 Lyon, Cedex 08. France). Twenty rat aortas were aseptically removed and preserved by freezing in liquid phase: The grafts were placed in a cryoprotective solution (DMSO) at varying concentrations and were subsequently frozen in a programmed fashion (temperature lowered at a rate of 1”Cimin and Kimin thereafter until a temperature of - 180°C was achieved). The frozen aortas were preserved in liquid nitrogen until used. These were thawed by simple heating to 37°C. A small segment was removed for histological observation by optical and electron microscopy and the remaining portion of the graft was implanted in rats of the same strain (IO isografts) or of different strains (IO allografts). The preserved grafts showed excellent conservation of the histological structure of the arterial walls (isolated endothelial lesions). The isografted vessels were all permeable after 20 days and 60% of these exhibited excellent histological aspects with respect to the allografts, the majority of the grafts showed important phasmocyte infiltration. These results suggest that this method of preservation is superior to those currently used for the conservation of blood vessels. Another long-term series of five isografted animals sacrificed between 90 to 255 days following the graft confirms the excellent evolution of the arterial grafts. This method of arterial cryopreservation is currently used for the realization of seven human arteriovenous fistulae for hemodialysis by using preserved femoral arteries obtained from cadavers.
716
ABSTRACTS,
2lst ANNUAL
G. M. ONIK,t w. K. HODDlCK,t AND BINSKY* (*Department of Mechanical
B. RUEngineering, University of California, Berkeley, California 94720and tDepartment of Radiology. University of California, San Francisco. California 94143). The use of low temperatures to destroy abnormal tissue (e.g., malignant tumors) is the basis for cryosurgical procedures. Cryosurgery would have the potential for treating some unresectable tumors in the liver, prostate, or brain were it not for the lack of a reliable means to monitor precisely the extent of the frozen region in real time. The use of ultrasound imaging technology has been proposed for this purpose.
MEETING
Experiments on transparent bovine gelatin samples were conducted to determine feasibility and accuracy of the method. A cryosurgical probe with a closed cylindrical tip was used to produce a cryolesion at one face of a rectangular solid gelatin sample. An ultrasound transducer was located at the opposite face to monitor the freezing process. A hemispherical frozen region in the gelatin was clearly defined in the ultrasonogram as a semicircular hyperechoic rim. The location of the change of phase interface is at the inner margin of this rim. Objects such as thermocouples placed in the gelatin prior to freezing, were visible in the ultrasonic image of the frozen region. Both 3.5 and 5 MHz ultrasound transducers were used. Size measurements of the frozen region correlated with the ultrasound image to within 0.7 mm.