- Email: [email protected]
The effect of freezing on the viability of rat heart muscle as measured by cytochrome oxidase activity
CRYOBIOLOGY Vol. 7, No. 4-6, 1971
THE EFFECT OF FREEZING ON THE VIABILITY OF RAT HEART MUSCLE AS MEASURED BY CYTOCHROME OXIDASE ACTIVITY L. J. RAMAZZOTTO, Department
of Physiology,
M. PLISKIN, Fairleigh
Dickinson
Recent experiments on the effects of extreme cold on cell and tissue integrity have shown that freezing and thawing often have a detrimental effect. Several theories have been advanced to explain the destructive mechanism.” 88118I4 It has been demonstrated that freezing and rewarming rates can affect cell and tissue viability.‘“, I’, I3 Still further, different cryoprotective agents, notably dimethyl sulfoxide, polyvinylpyrrolidone, glycerol, lipid compounds, etc., can alter the over-all freezing and thawing effects.‘, ‘* 12,*’ Studies have been done on in vitro and in viva freezing of whole organs. One organ often used is the heart since the cellular makeup is of one basic type (myocardial tissue) and it is easily perfused by means of the great vessels leading to it .I* ’ A measurement of recovery after freezethaw that is often used with heart muscle is contractile ability.“, I5 Some studies have been done on enzyme function in frozen-thawed heart muscle, mainly studies of glutamic-oxalacetic transaminaseP rlt the same time, enzyme activity studies have been done on solutions of purified enzymes after freeze-thaw,’ and on enz,yme activity in frozen tissues, usually other than heart muscle.’ Enzyme assays and activity studies have been run on normal unfrozen heart muscle. Some studies have been done on the succinate dehydrogenase system in heart muscle.’ These experiments on the activity of the cytochrome oxidase system were used as a measure of recovery of heart muscle tissue after quickfreezing in liquid nitrogen and subsequent thawing out. This paper is a preliminary report dealing with the effects of freeze-thaw treatment on the cytochrome system in two differently prepared samples of heart muscle tissue. Received April 13,197O.
AND
E. ROCHVARG
University,
Teaneck, New Jersey
MATERIALS
AND
METHODS
The assay process for the activity of the cytochrome oxidase system is that of Cooperstein and Lazzara (1951). The buffer and solution strengths were as follows: 0.03 M phosphate buffer. For the original homogenization the tissue was 1: 16.6 of the solution and was diluted out to be 1:1,666 of it. The final dilution of the tissue, for the spectrophotometric readings, was 1: 10,000. Obtaining tissue. Rat hearts were excised from white laboratory rats which had been decapitated. The hearts were homogenized in a 1: 16.6 ratio of cell to phosphate buffer in a VirTis 23 at full speed (approximately 23,000 rpm) for 1% min. This was then transferred to a Teflon and glass tissue homogenizer for 2 min. Controls were prepared in the same way. Freezing and thawing of tissue. Rat heart muscle, prepared as described, was placed in a 2-dram glass vial and frozen in liquid nitrogen (-320°F) for 30 min in the phosphate buffer solution in a 1: 16.6 dilution. The samples were thawed in a 37°C water bath until the buffer suspension was liquid. Controls were homogenized and allowed to stand at room temperature for 30 min and the length of time for the thawing process. Measurement
of cytochrome
oxidase
activity.
The samples obtained as described above were diluted to 1: 1,666 with phosphate buffer. Fivetenths milliliter of this solution was added to 2.5 ml 0.05% reduced cytochrome c solution (this gave a 1: 10,000 dilution). This was thoroughly mixed and readings were taken on a DB-G spectrophotometer on O.D. at 1-min intervals for 5 min. Immediately after the final reading, 0.1 ml 0.01 M potassium ferrocyanide was added to oxidize the cytochrome c completely and a last reading was taken. Another set of trials was run, in which the heart muscle was frozen in 1 cm3 and then 256
VIABILITY
OF RAT
HEART
MUSCLE
thawed and homogenized in the same manner as before. Likewise, cyt’ochrome oxidase activity was measured in the same way.
TABLE
A log (Cy i- Fe++) min
1
“o&pI
RESULTS
The results were calculated following equation :
257
according to the
s (Da - Do, - log (D,z - Do,) h - fz
where D,, is O.D. at zero time, D,, is O.D. after 5 min, and D,. is O.D. after the addition of potassium ferrocyanide. The results are summarized in Table 1. The readings obtained are in terms of: Group I, the control group; Group II, the group that, was homogenized and then frozen; and Group III, the group that was frozen in cubic centimeter pieces and then homogcnized. DISCUSSION
‘The same control was used for both sets of experiments, since nothing was done to the control beyond homogenizing it. Thus, one set of homogenized controls gave a basis for comparison between two different freezing methods and their respective effects on the cgtochrome oxidase system of rat, heart muscle cells. As can be seen from the table, t’he per cent of cytochrome oxidase act)ivity as compared to that of the control was greater in the samples frozen whole and then homogenized (79.35%). It was much less in the samples homogenized first and then frozen (48.52%). These results seem to bear out some of the theorirs expressed about the mechanism of freeze-thaw damage to cells.“’ a. ‘I* I’ That is, in the homogenized and frozen samples, more surface area, and therefore more cells, was open to damage from exposure to ice crystals, and necrosis due to such exposure. However, in the whole frozen and homogenized samples, only those cells on the outermost borders were open to ice crystal damage and resulting necrosis. The large majority of cells in these samples were within the sample and thus were protected. These could easily account for 79.35% of maximum activity. However, before this could be advanced as a theory, it would be necessary to do further studies, some of which would involve the making of serial slides of pieces of heart muscle frozen whole and then thawed. Such slides would show
Average
Mean Percentage
of
0.0432 0.0402 100.007,
Group II 11
Gro% II1
0.0219
0.0319
0.0229 48.527,
0.0289 79.35%
control
the degree of necrosis present from the front of the piece to the back and would indicate the validity of the hypothesis. Further studies along this line are planned, as well as studies of the succinate dehydrogenase activity in rat heart muscle. This points to the great difficulty in freezing studies: one enzyme system may be affected little or not at all, but another or several others may be seriously affected. There are so many systems involved that it seems to be almost impossible to arrive at freezing methods generally applicable. At the same time, in freezing large pieces of tissue (and whole organs), the extent of recovery may be great as compared to normal controls, but there is no way, as yet, of determining if even this is compatible with life (as applied to the idea of organ banks). SUMMARY
Rat heart muscle was studied for cytochrome oxidase activity after freezing, according to the method of Cooperstein and Lazzara. Two methods of freezing were used: the muscle was homogenized and then frozen for 30 min in one set of experiment’s; in the other set the muscle was frozen in cubic centimeter pieces and homogenized after thawing. Both were thawed in a 37°C water bath until the buffer suspension became liquid. The former set of experiments showed cytochrome oxidase activity to be 48.52% of the control. The latter set showed activity of 79.35% of the control. These results are probably best explained on a basis of protection from ice crystal damage and resulting necrosis. REFERENCES 1. Athreya,
B. H., Coriell, L. L., Greene, A. E., and Lehr, H. B. In vitro preservation of functioning rabbit hearts in a depolarized state at 4°C. Cryobiology, 6’: 403-409, 1969. 2. Doebbler, G. F. Cryoprotective compounds: Review and discussion of structure and function. Cryobiology, S: 2-11, 1966.
258
RAMAZZOTTO,
PLISKIN,
3. Fishbein, W. N.. and Stowell, R. E. Studies on the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay. I. Temporal localization of the injury phase during slow freezing. Cryobiology, 4: 283289, 1968. 4. Garzon, A. A., Kornberg, E., Pangan, J., Stuckey. J. H., and Karlson, K. E. Myocardial contractilitv after 24 hours of hvpothermic (4°C) storage. Cryobiology, 6: “347-351, 1969. 5. Greiff, D., and Kelly. R. T. Cryotolerance of enzymes. I. Freezing of lactic dehydrogenase. Cryobiology, 2: 335341,1966. 6. Hechter, 0. Role of water structure in the molecular organization of cell membranes. Fed. Proc., 24 (Suppl.) : SSl-SlO2, 1965. 7. Hulfner, M., Buckley, L., and Hollecher, T. Studies on the exchange of hydrogen between succinate and water as catalyzed by (beef) heart muscle succinic dehydrogenase. Biothem. Biophvs. Res. Commun. 28: 791-796. 1967. . 8. Karow. A. M.. and Webb, W. R. Tissue freezing: A theory for injury and survival. Cryobiology, 2: 99108.1965.
AND ROCHVARG
9. Klein, E., Lyman, R. B., Peterson, L., Berger, R. I., and Smith, G. K. The effects of dimethylsulfoxide on lipoproteins stored at low temperatures. Cryobiology, 3: 328-334, 1967. 10. Mazur, P. Theoretical and experimental effects of cooling and warming velocity on the survival of frozen and thawed cells. Cryobiology, 2: 181-192, 1966. 11. Mazur, P. Causes of injury in frozen and thawed cells. Fed. Proc. 24: No. 2, Part III: S175-S182,1965. 12. Mazur, P.. Farrant, J., Leibo, S. P., and Chu, E. H. Y. Survival of hamster tissue culture cells after freezing and thawing. Interaction between protective solutes and cooling and warming rates. Cryobiology, 6: l-9,1969. 13. Meryman, H. T. The interpretation of freezing rates in biological materials. Cryobiology, 2: 165-170, 1966. 14. Meryman, H. T. General principles of freezing and freezing injury in cellular materials. Ann. N. Y. Acad. Sci., 85: 503-509. 1960. 15. SchGpf-Ebner, E.,’ Gross, W. ‘O., and Bucher, 0. M. Pulsatile activity of isolated heart muscle cells after freezing storage. Cryobiology, 4: 200-203, 1968.
THE EFFECT OF FREEZING ON THE VIABILITY OF RAT HEART MUSCLE AS MEASURED BY CYTOCHROME OXIDASE ACTIVITY L. J. RAMAZZOTTO, Department
of Physiology,
M. PLISKIN, Fairleigh
Dickinson
Recent experiments on the effects of extreme cold on cell and tissue integrity have shown that freezing and thawing often have a detrimental effect. Several theories have been advanced to explain the destructive mechanism.” 88118I4 It has been demonstrated that freezing and rewarming rates can affect cell and tissue viability.‘“, I’, I3 Still further, different cryoprotective agents, notably dimethyl sulfoxide, polyvinylpyrrolidone, glycerol, lipid compounds, etc., can alter the over-all freezing and thawing effects.‘, ‘* 12,*’ Studies have been done on in vitro and in viva freezing of whole organs. One organ often used is the heart since the cellular makeup is of one basic type (myocardial tissue) and it is easily perfused by means of the great vessels leading to it .I* ’ A measurement of recovery after freezethaw that is often used with heart muscle is contractile ability.“, I5 Some studies have been done on enzyme function in frozen-thawed heart muscle, mainly studies of glutamic-oxalacetic transaminaseP rlt the same time, enzyme activity studies have been done on solutions of purified enzymes after freeze-thaw,’ and on enz,yme activity in frozen tissues, usually other than heart muscle.’ Enzyme assays and activity studies have been run on normal unfrozen heart muscle. Some studies have been done on the succinate dehydrogenase system in heart muscle.’ These experiments on the activity of the cytochrome oxidase system were used as a measure of recovery of heart muscle tissue after quickfreezing in liquid nitrogen and subsequent thawing out. This paper is a preliminary report dealing with the effects of freeze-thaw treatment on the cytochrome system in two differently prepared samples of heart muscle tissue. Received April 13,197O.
AND
E. ROCHVARG
University,
Teaneck, New Jersey
MATERIALS
AND
METHODS
The assay process for the activity of the cytochrome oxidase system is that of Cooperstein and Lazzara (1951). The buffer and solution strengths were as follows: 0.03 M phosphate buffer. For the original homogenization the tissue was 1: 16.6 of the solution and was diluted out to be 1:1,666 of it. The final dilution of the tissue, for the spectrophotometric readings, was 1: 10,000. Obtaining tissue. Rat hearts were excised from white laboratory rats which had been decapitated. The hearts were homogenized in a 1: 16.6 ratio of cell to phosphate buffer in a VirTis 23 at full speed (approximately 23,000 rpm) for 1% min. This was then transferred to a Teflon and glass tissue homogenizer for 2 min. Controls were prepared in the same way. Freezing and thawing of tissue. Rat heart muscle, prepared as described, was placed in a 2-dram glass vial and frozen in liquid nitrogen (-320°F) for 30 min in the phosphate buffer solution in a 1: 16.6 dilution. The samples were thawed in a 37°C water bath until the buffer suspension was liquid. Controls were homogenized and allowed to stand at room temperature for 30 min and the length of time for the thawing process. Measurement
of cytochrome
oxidase
activity.
The samples obtained as described above were diluted to 1: 1,666 with phosphate buffer. Fivetenths milliliter of this solution was added to 2.5 ml 0.05% reduced cytochrome c solution (this gave a 1: 10,000 dilution). This was thoroughly mixed and readings were taken on a DB-G spectrophotometer on O.D. at 1-min intervals for 5 min. Immediately after the final reading, 0.1 ml 0.01 M potassium ferrocyanide was added to oxidize the cytochrome c completely and a last reading was taken. Another set of trials was run, in which the heart muscle was frozen in 1 cm3 and then 256
VIABILITY
OF RAT
HEART
MUSCLE
thawed and homogenized in the same manner as before. Likewise, cyt’ochrome oxidase activity was measured in the same way.
TABLE
A log (Cy i- Fe++) min
1
“o&pI
RESULTS
The results were calculated following equation :
257
according to the
s (Da - Do, - log (D,z - Do,) h - fz
where D,, is O.D. at zero time, D,, is O.D. after 5 min, and D,. is O.D. after the addition of potassium ferrocyanide. The results are summarized in Table 1. The readings obtained are in terms of: Group I, the control group; Group II, the group that, was homogenized and then frozen; and Group III, the group that was frozen in cubic centimeter pieces and then homogcnized. DISCUSSION
‘The same control was used for both sets of experiments, since nothing was done to the control beyond homogenizing it. Thus, one set of homogenized controls gave a basis for comparison between two different freezing methods and their respective effects on the cgtochrome oxidase system of rat, heart muscle cells. As can be seen from the table, t’he per cent of cytochrome oxidase act)ivity as compared to that of the control was greater in the samples frozen whole and then homogenized (79.35%). It was much less in the samples homogenized first and then frozen (48.52%). These results seem to bear out some of the theorirs expressed about the mechanism of freeze-thaw damage to cells.“’ a. ‘I* I’ That is, in the homogenized and frozen samples, more surface area, and therefore more cells, was open to damage from exposure to ice crystals, and necrosis due to such exposure. However, in the whole frozen and homogenized samples, only those cells on the outermost borders were open to ice crystal damage and resulting necrosis. The large majority of cells in these samples were within the sample and thus were protected. These could easily account for 79.35% of maximum activity. However, before this could be advanced as a theory, it would be necessary to do further studies, some of which would involve the making of serial slides of pieces of heart muscle frozen whole and then thawed. Such slides would show
Average
Mean Percentage
of
0.0432 0.0402 100.007,
Group II 11
Gro% II1
0.0219
0.0319
0.0229 48.527,
0.0289 79.35%
control
the degree of necrosis present from the front of the piece to the back and would indicate the validity of the hypothesis. Further studies along this line are planned, as well as studies of the succinate dehydrogenase activity in rat heart muscle. This points to the great difficulty in freezing studies: one enzyme system may be affected little or not at all, but another or several others may be seriously affected. There are so many systems involved that it seems to be almost impossible to arrive at freezing methods generally applicable. At the same time, in freezing large pieces of tissue (and whole organs), the extent of recovery may be great as compared to normal controls, but there is no way, as yet, of determining if even this is compatible with life (as applied to the idea of organ banks). SUMMARY
Rat heart muscle was studied for cytochrome oxidase activity after freezing, according to the method of Cooperstein and Lazzara. Two methods of freezing were used: the muscle was homogenized and then frozen for 30 min in one set of experiment’s; in the other set the muscle was frozen in cubic centimeter pieces and homogenized after thawing. Both were thawed in a 37°C water bath until the buffer suspension became liquid. The former set of experiments showed cytochrome oxidase activity to be 48.52% of the control. The latter set showed activity of 79.35% of the control. These results are probably best explained on a basis of protection from ice crystal damage and resulting necrosis. REFERENCES 1. Athreya,
B. H., Coriell, L. L., Greene, A. E., and Lehr, H. B. In vitro preservation of functioning rabbit hearts in a depolarized state at 4°C. Cryobiology, 6’: 403-409, 1969. 2. Doebbler, G. F. Cryoprotective compounds: Review and discussion of structure and function. Cryobiology, S: 2-11, 1966.
258
RAMAZZOTTO,
PLISKIN,
3. Fishbein, W. N.. and Stowell, R. E. Studies on the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay. I. Temporal localization of the injury phase during slow freezing. Cryobiology, 4: 283289, 1968. 4. Garzon, A. A., Kornberg, E., Pangan, J., Stuckey. J. H., and Karlson, K. E. Myocardial contractilitv after 24 hours of hvpothermic (4°C) storage. Cryobiology, 6: “347-351, 1969. 5. Greiff, D., and Kelly. R. T. Cryotolerance of enzymes. I. Freezing of lactic dehydrogenase. Cryobiology, 2: 335341,1966. 6. Hechter, 0. Role of water structure in the molecular organization of cell membranes. Fed. Proc., 24 (Suppl.) : SSl-SlO2, 1965. 7. Hulfner, M., Buckley, L., and Hollecher, T. Studies on the exchange of hydrogen between succinate and water as catalyzed by (beef) heart muscle succinic dehydrogenase. Biothem. Biophvs. Res. Commun. 28: 791-796. 1967. . 8. Karow. A. M.. and Webb, W. R. Tissue freezing: A theory for injury and survival. Cryobiology, 2: 99108.1965.
AND ROCHVARG
9. Klein, E., Lyman, R. B., Peterson, L., Berger, R. I., and Smith, G. K. The effects of dimethylsulfoxide on lipoproteins stored at low temperatures. Cryobiology, 3: 328-334, 1967. 10. Mazur, P. Theoretical and experimental effects of cooling and warming velocity on the survival of frozen and thawed cells. Cryobiology, 2: 181-192, 1966. 11. Mazur, P. Causes of injury in frozen and thawed cells. Fed. Proc. 24: No. 2, Part III: S175-S182,1965. 12. Mazur, P.. Farrant, J., Leibo, S. P., and Chu, E. H. Y. Survival of hamster tissue culture cells after freezing and thawing. Interaction between protective solutes and cooling and warming rates. Cryobiology, 6: l-9,1969. 13. Meryman, H. T. The interpretation of freezing rates in biological materials. Cryobiology, 2: 165-170, 1966. 14. Meryman, H. T. General principles of freezing and freezing injury in cellular materials. Ann. N. Y. Acad. Sci., 85: 503-509. 1960. 15. SchGpf-Ebner, E.,’ Gross, W. ‘O., and Bucher, 0. M. Pulsatile activity of isolated heart muscle cells after freezing storage. Cryobiology, 4: 200-203, 1968.