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Effect of DMSO on the dielectric properties of canine kidney tissue
CHYOBIOLOG’Y
13, 581-585
(1976)
BRIEF COMMUNICATION Effect
of DMSO on the Dielectric KENNETH
R. FOSTER,?
Properties
of Canine
R. THOMAS BELL, III,3 RICHARD AND BOHDAN DENYSYKS
Kidney Tissue1
WHITTINGTON,s
2 Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20014, and 3 Naval Medical Research Institute, Bethesda, Maryland 20014
One major difficulty with attempts to prolong the viability of solid organs by freezing i,s the need to rapidly and uniformly thaw the tissue after storage. An attractive approach-using a commercial microwave oven operating at 2450 or 915 MHz-has had only limited success, partially because of the nonuniform heating pattern produced by the microwave field (1, 2, 5). To ascertain the probable effect of a commonly used cryoprotective agent, dimethyl sulfoxide (DMSO), on the electromagnetic heating process, we measured the conductivity and permittivity of canine kidney tissue over the temperature range -20°C to +2O”C, and over the frequency range 2 to 900 MHz. Most of the kidneys were perfused with canine plasma contain-
ing 10% (by volume) DMSO (with addition of NaCl to maintain a constant ionic strength in the solution) ; others were perfused with physiological saline only. The big increase in tissue conductivity between -20°C and 0°C is the underlying reason for the unstable heating of the kidneys by a microwave field. MATERIALS
METHODS
Kidneys, weighing 60 to SO g, were removed from adult male mongrel dogs by laparotomy incision. Anesthesia was induced with sodium pentobarbital and later maintained with a mixture of halothane, nitrous oxide, and oxygen. The surgical procedures were done in connection with an immunological study on renal rejection, requiring autotransplantation of one kidney and removal of the contralateral organ, and are described in detail elsewhere (3). The removed kidneys were perfused either with canine plasma containing DMSO on a Belzer perfusion apparatus or with gravityinfused physiological saline, and then stored at -10°C in a standard freezer until experiment’ally used. Samples of the DMSOperfused tissue were analyzed by tritium label distribution studies to verify that the DMSO had uniformly penetrated the tissue. Samples of approximately 1 cm3 volume
Received November 19, 1975. 1 This work was supported by the Navy Research and Development Command, Research Task No. MR041.02.01.0024. The opinions or assertions contained hferein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The animals used in this study were handled in accordance with the provisions of Public Law 89-44 as amended by Public Law 91-579, the “Animal Welfare Act of 1970” and the principles outlined in the “Guide for the Care and Use of Laboratory Animals,” U. S. Department of Health, Education and Welfare Publication No. (NIH) 72-23. 581 Copyright 1976 by Academic Press, Inc. All rights o3 reproduction in any form reserved.
AND
582
BRIEF
COMMUNICATION
FIG. 1. (A) The specific conductivity, q of saline- and DMSO-perfused canine kidney tissue as a function of temperature. The conductivities of cortical and medullary tissues were the same to within data reproducibility and are included together. The SEM for the data at -20°C is indicated; the remaining data at higher temperatures have somewhat smaller relative uncertainties. (B) The specific conductivity of 0.15 N NaCl and 0.15 N NaCl-10% DMSO mixtures (v/v) as a function of temperature. The similarity between A and B shows that the DMSO-induced increase in the conductivity of frozen tissue results from a colligative property of the DMSO-electrolyte solution.
were removed from the cortex and medulla, and packed into a stainless steel and Teflon dielectric cell constructed from #alength of 50 ohm coaxial air line, as described elsewhere (6). The sample temperature was controlled to within 1°C by delivering cold nitrogen gas through a coaxial jacket surrounding the cell. Measurements over the frequency range of 3-100 MHz were performed using a Booton R-X meter and a Wayne-Kerr model 801 admittance bridge. At 500 MHz, limited data on frozen tissue were obtained from this cell using a General Radio model 1607 U.H.F. admittance bridge and correcting for the cell’s series inductance of 10ms H. Data on somewhat larger samples of thawed tissue were obtained at 900 MHz by measuring the input admittance of a thermostatically controlled
5.75 cm length of coaxial air line using the admittance bridge. To within the reproducibility of the data, the dielectric properties of the cortex and medulla are identical, and the results are included together in the discussion below. Several successive freeze-thaw cycles produced no change in the measured dielectric properties. Our measurements on saline perfused tissue at 20°C agree well with those reported by Schwan (7) for (human) kidney tissue. Conductivity measurements were performed on 0.15 N NaCl and saline-10% DMSO (v/v) mixtures using a platinized conductivity cell of standard design and a Wayne-Kerr model 601 impedance bridge operating at 200 kHz. The observed conductivities of these two solutions were inde-
BRIEF
01 ’
COMMUNICATION
10
I
I
’
“I
5s3
100
1000
1, MHz
FIG. 51.The measured of frequency. Data from son. DMSO substantially tissue, probably leading pendent of frequency as expected.
specific conductivity, g, of DMSO-perfused kidney tissue as a function saline-perfused kidneys at 10°C and -20°C are shown for comparireduces the difference in conductivity between “frozen” and “thawed” to more uniform thawing in a microwave field.
at all temperatures,
the tissue conductivity at temperatures below -10°C is constant over the frequency range of 3 to 500 MHz; that of the thawed tissue increases twofold (saline-perfused) RESULTS AND DISCUSSION to four fold (DMSO-perfused) from 3 to The property which is most irnportant- in 900 MHz. At 900 MHz, the conductivities determiniqg the electromagnetic heating of the DMSO-perfused and saline-perfused behavior of the tissue is c, the specific con- organs are roughly the same. The permittivductivity of the tissue. Figure 1A shows the ity e’ of the DMSO-perfused tissue is shown conductivity at 10 MHz of DMSO-perfused in Fig. 3, with that of the saline-perfused and saline-perfused organs. Each point tissue at -20°C and +1O”C shown for represents the ‘average of six measurements comparison. Again, DMSO significantly reon three DMSO-perfused organs, or of four duces the change in the dielectric propmeasurements on two saline-perfused kiderties of the tissue as it is thawed. Because neys. The SEM is approximately 25% exof equipment limitations, measurements cept at -20°C when it increases to nearly were not performed at 2410 MHz, but these 100% because of the lower values of u. DMSO-produced changes in the dielectric At -20°C DMSO increases the conductivproperties of kidney would be expected to ity of the tj.ssue by nearly fivefold; at 20°C occur at higher frequencies as well. it decreases the conductivity by 40%. These Unfortunately, we cannot realistically effects are observed in DMSO-saline SO~Umodel the dielectric heating patterns in a tions #aswell (Fig. 1B). Figure 2 shows that
584
BRIEF
COMMUNICATION
FIG. 3. Relative permittivity, E’, of DMSO-perfused kidney tissue at 10°C and -20°C are included for comparison.
partially thawed organ irradiated by a microwave oven. However, detailed calculations, concentric tissue assuming spheres whose dielectric properties are characteristic of frozen and thawed tissue irradiated with plane electromagnetic waves, show that the greatest nonuniformity in the dissipated power occurs at the interface between the two sections (4). This nonuniformity results from the mathematical boundary condition that the tangential component of the electric field, Et, be continuous across the interface between the thawed and frozen sections. The power deposited by this field component is proportional to aEct2. To improve the uniformity of the heating, it therefore appears most important to reduce the difference in the conductivities between the “frozen” and the “thawed” tissue. Since the conductivity of the “thawed” tissue, unlike that of the “frozen” tissue, rises with frequency above 100 MHz, our data suggest that more uniform heating might be obtained using frequencies lower than 915 or 2450 MHZ. Our results show ‘a significant effect of the cryoprotectant DMSO on the conductivity of canine kidney tissue. Presumably,
tissue. Data from
saline-perfused
this will substantially improve the microwave heating pattern in frozen tissues. This effect, in addition to pharmacological considerations, should be a factor in the choice of a cryoprotective agent for solid organs which are to be thawed by radiofrequency or microwave irradiation. SUMMARY
The dielectric permittivity and conductivity of canine kidney tissue samples were measured at R, frequencies between -20°C and +2O”C. Some of the kidneys had been perfused with DMSO ( 10% ) in canine plasma, others with physiological saline alone. The DMSO greatly increases the conductivity of frozen tissue above that of tissue not treated with this cryoprotectant. Apparently, the chief reason for nonuniform heating ‘of a partially frozen organ in a microwave field is the great ‘change in tissue conductivity as it thaws. We suggest that the effect on the conductivity of tissue should be considered in the choice of a cryoprotectant for tissues which are to be thawed by microwave or radiofrequency irradiation.
BRIEF
COMMUNICATION
ACKNOWLEDGMENT We thank Dr. Henry S. Ho, Division of Biological Effects, Bureau of Radiological Health, Food and Drug Administration, United States Public Health Service, Rockville, Maryland, for discussions and for preliminary calculations modeling the microwave thawing of frozen tissues.
REFERENCES 1. Burns, C. I?., Burdett, E. C., and Karow, A. M. Thawing of rabbit kidneys from -79°C with 2450 MHz radiation (Abstr.), Society for Cryobiology, Twelfth Annual Meeting, 1975. CyobioZogy 12, 577 ( 1975). 2. Dietzman, R. H., Rebelo, A. E., Graham, E. F., Crabo, 13. G., and Lillehei, R. C. Long term
3.
4. 5.
6.
7.
585
functional success following freezing of canine kidneys. Surgery 74, 181-189 (1973). Filo, R. S., Dickson, L. G., Suba, E. A., and Sell, K. W. Immunologic injury induced by ex oizjo perfusion of canine renal autographs. Surgery 76, 88-100 ( 1974). Ho, H. S., personal communication. Lehr, A. B., Berggren, R. B., Summers, A. L., and Lotke, P. A. Freezing and thawing of large organs. Cryobiology 1, 194-197 ( 1964). O’Konski, C. T., and Edwards, A. Cell for dielectric measurements in the high radio frequency region. Rezj. Sci. In&. 39, 1456-1458 (1968). Schwan, H. P. Determination of biological impedances. In “Physical Techniques in Biological Research” ( W. L. Nastuk, Ed.), Vol. 6, pp. 323-407. Academic Press, New York, 1963.
13, 581-585
(1976)
BRIEF COMMUNICATION Effect
of DMSO on the Dielectric KENNETH
R. FOSTER,?
Properties
of Canine
R. THOMAS BELL, III,3 RICHARD AND BOHDAN DENYSYKS
Kidney Tissue1
WHITTINGTON,s
2 Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20014, and 3 Naval Medical Research Institute, Bethesda, Maryland 20014
One major difficulty with attempts to prolong the viability of solid organs by freezing i,s the need to rapidly and uniformly thaw the tissue after storage. An attractive approach-using a commercial microwave oven operating at 2450 or 915 MHz-has had only limited success, partially because of the nonuniform heating pattern produced by the microwave field (1, 2, 5). To ascertain the probable effect of a commonly used cryoprotective agent, dimethyl sulfoxide (DMSO), on the electromagnetic heating process, we measured the conductivity and permittivity of canine kidney tissue over the temperature range -20°C to +2O”C, and over the frequency range 2 to 900 MHz. Most of the kidneys were perfused with canine plasma contain-
ing 10% (by volume) DMSO (with addition of NaCl to maintain a constant ionic strength in the solution) ; others were perfused with physiological saline only. The big increase in tissue conductivity between -20°C and 0°C is the underlying reason for the unstable heating of the kidneys by a microwave field. MATERIALS
METHODS
Kidneys, weighing 60 to SO g, were removed from adult male mongrel dogs by laparotomy incision. Anesthesia was induced with sodium pentobarbital and later maintained with a mixture of halothane, nitrous oxide, and oxygen. The surgical procedures were done in connection with an immunological study on renal rejection, requiring autotransplantation of one kidney and removal of the contralateral organ, and are described in detail elsewhere (3). The removed kidneys were perfused either with canine plasma containing DMSO on a Belzer perfusion apparatus or with gravityinfused physiological saline, and then stored at -10°C in a standard freezer until experiment’ally used. Samples of the DMSOperfused tissue were analyzed by tritium label distribution studies to verify that the DMSO had uniformly penetrated the tissue. Samples of approximately 1 cm3 volume
Received November 19, 1975. 1 This work was supported by the Navy Research and Development Command, Research Task No. MR041.02.01.0024. The opinions or assertions contained hferein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The animals used in this study were handled in accordance with the provisions of Public Law 89-44 as amended by Public Law 91-579, the “Animal Welfare Act of 1970” and the principles outlined in the “Guide for the Care and Use of Laboratory Animals,” U. S. Department of Health, Education and Welfare Publication No. (NIH) 72-23. 581 Copyright 1976 by Academic Press, Inc. All rights o3 reproduction in any form reserved.
AND
582
BRIEF
COMMUNICATION
FIG. 1. (A) The specific conductivity, q of saline- and DMSO-perfused canine kidney tissue as a function of temperature. The conductivities of cortical and medullary tissues were the same to within data reproducibility and are included together. The SEM for the data at -20°C is indicated; the remaining data at higher temperatures have somewhat smaller relative uncertainties. (B) The specific conductivity of 0.15 N NaCl and 0.15 N NaCl-10% DMSO mixtures (v/v) as a function of temperature. The similarity between A and B shows that the DMSO-induced increase in the conductivity of frozen tissue results from a colligative property of the DMSO-electrolyte solution.
were removed from the cortex and medulla, and packed into a stainless steel and Teflon dielectric cell constructed from #alength of 50 ohm coaxial air line, as described elsewhere (6). The sample temperature was controlled to within 1°C by delivering cold nitrogen gas through a coaxial jacket surrounding the cell. Measurements over the frequency range of 3-100 MHz were performed using a Booton R-X meter and a Wayne-Kerr model 801 admittance bridge. At 500 MHz, limited data on frozen tissue were obtained from this cell using a General Radio model 1607 U.H.F. admittance bridge and correcting for the cell’s series inductance of 10ms H. Data on somewhat larger samples of thawed tissue were obtained at 900 MHz by measuring the input admittance of a thermostatically controlled
5.75 cm length of coaxial air line using the admittance bridge. To within the reproducibility of the data, the dielectric properties of the cortex and medulla are identical, and the results are included together in the discussion below. Several successive freeze-thaw cycles produced no change in the measured dielectric properties. Our measurements on saline perfused tissue at 20°C agree well with those reported by Schwan (7) for (human) kidney tissue. Conductivity measurements were performed on 0.15 N NaCl and saline-10% DMSO (v/v) mixtures using a platinized conductivity cell of standard design and a Wayne-Kerr model 601 impedance bridge operating at 200 kHz. The observed conductivities of these two solutions were inde-
BRIEF
01 ’
COMMUNICATION
10
I
I
’
“I
5s3
100
1000
1, MHz
FIG. 51.The measured of frequency. Data from son. DMSO substantially tissue, probably leading pendent of frequency as expected.
specific conductivity, g, of DMSO-perfused kidney tissue as a function saline-perfused kidneys at 10°C and -20°C are shown for comparireduces the difference in conductivity between “frozen” and “thawed” to more uniform thawing in a microwave field.
at all temperatures,
the tissue conductivity at temperatures below -10°C is constant over the frequency range of 3 to 500 MHz; that of the thawed tissue increases twofold (saline-perfused) RESULTS AND DISCUSSION to four fold (DMSO-perfused) from 3 to The property which is most irnportant- in 900 MHz. At 900 MHz, the conductivities determiniqg the electromagnetic heating of the DMSO-perfused and saline-perfused behavior of the tissue is c, the specific con- organs are roughly the same. The permittivductivity of the tissue. Figure 1A shows the ity e’ of the DMSO-perfused tissue is shown conductivity at 10 MHz of DMSO-perfused in Fig. 3, with that of the saline-perfused and saline-perfused organs. Each point tissue at -20°C and +1O”C shown for represents the ‘average of six measurements comparison. Again, DMSO significantly reon three DMSO-perfused organs, or of four duces the change in the dielectric propmeasurements on two saline-perfused kiderties of the tissue as it is thawed. Because neys. The SEM is approximately 25% exof equipment limitations, measurements cept at -20°C when it increases to nearly were not performed at 2410 MHz, but these 100% because of the lower values of u. DMSO-produced changes in the dielectric At -20°C DMSO increases the conductivproperties of kidney would be expected to ity of the tj.ssue by nearly fivefold; at 20°C occur at higher frequencies as well. it decreases the conductivity by 40%. These Unfortunately, we cannot realistically effects are observed in DMSO-saline SO~Umodel the dielectric heating patterns in a tions #aswell (Fig. 1B). Figure 2 shows that
584
BRIEF
COMMUNICATION
FIG. 3. Relative permittivity, E’, of DMSO-perfused kidney tissue at 10°C and -20°C are included for comparison.
partially thawed organ irradiated by a microwave oven. However, detailed calculations, concentric tissue assuming spheres whose dielectric properties are characteristic of frozen and thawed tissue irradiated with plane electromagnetic waves, show that the greatest nonuniformity in the dissipated power occurs at the interface between the two sections (4). This nonuniformity results from the mathematical boundary condition that the tangential component of the electric field, Et, be continuous across the interface between the thawed and frozen sections. The power deposited by this field component is proportional to aEct2. To improve the uniformity of the heating, it therefore appears most important to reduce the difference in the conductivities between the “frozen” and the “thawed” tissue. Since the conductivity of the “thawed” tissue, unlike that of the “frozen” tissue, rises with frequency above 100 MHz, our data suggest that more uniform heating might be obtained using frequencies lower than 915 or 2450 MHZ. Our results show ‘a significant effect of the cryoprotectant DMSO on the conductivity of canine kidney tissue. Presumably,
tissue. Data from
saline-perfused
this will substantially improve the microwave heating pattern in frozen tissues. This effect, in addition to pharmacological considerations, should be a factor in the choice of a cryoprotective agent for solid organs which are to be thawed by radiofrequency or microwave irradiation. SUMMARY
The dielectric permittivity and conductivity of canine kidney tissue samples were measured at R, frequencies between -20°C and +2O”C. Some of the kidneys had been perfused with DMSO ( 10% ) in canine plasma, others with physiological saline alone. The DMSO greatly increases the conductivity of frozen tissue above that of tissue not treated with this cryoprotectant. Apparently, the chief reason for nonuniform heating ‘of a partially frozen organ in a microwave field is the great ‘change in tissue conductivity as it thaws. We suggest that the effect on the conductivity of tissue should be considered in the choice of a cryoprotectant for tissues which are to be thawed by microwave or radiofrequency irradiation.
BRIEF
COMMUNICATION
ACKNOWLEDGMENT We thank Dr. Henry S. Ho, Division of Biological Effects, Bureau of Radiological Health, Food and Drug Administration, United States Public Health Service, Rockville, Maryland, for discussions and for preliminary calculations modeling the microwave thawing of frozen tissues.
REFERENCES 1. Burns, C. I?., Burdett, E. C., and Karow, A. M. Thawing of rabbit kidneys from -79°C with 2450 MHz radiation (Abstr.), Society for Cryobiology, Twelfth Annual Meeting, 1975. CyobioZogy 12, 577 ( 1975). 2. Dietzman, R. H., Rebelo, A. E., Graham, E. F., Crabo, 13. G., and Lillehei, R. C. Long term
3.
4. 5.
6.
7.
585
functional success following freezing of canine kidneys. Surgery 74, 181-189 (1973). Filo, R. S., Dickson, L. G., Suba, E. A., and Sell, K. W. Immunologic injury induced by ex oizjo perfusion of canine renal autographs. Surgery 76, 88-100 ( 1974). Ho, H. S., personal communication. Lehr, A. B., Berggren, R. B., Summers, A. L., and Lotke, P. A. Freezing and thawing of large organs. Cryobiology 1, 194-197 ( 1964). O’Konski, C. T., and Edwards, A. Cell for dielectric measurements in the high radio frequency region. Rezj. Sci. In&. 39, 1456-1458 (1968). Schwan, H. P. Determination of biological impedances. In “Physical Techniques in Biological Research” ( W. L. Nastuk, Ed.), Vol. 6, pp. 323-407. Academic Press, New York, 1963.