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Studies on the preservation of canine lung at subzero temperatures
Studies on the preservation of canine lung at subzero temperatures Gunji Okaniwa, M.D., Tasuku Nakada, M.D., Minoru Kawakami, M.D., Shigejumi Fujimura, M.D., Yoshikazu Arakaki, M.D., Shigehiro Chiba, M.D., Minoru Yonechi, M.D., Yoshitoshi Kagami, M.D., and Chigashi Suzuki, M.D., * Sendai, Japan
There have been two approaches to the problem of organ preservation; one is storage above the freezing point of water and the other is storage below the freezing point. The extensive cell damage produced by freezing and warming of large tissue masses cannot be totally avoided. However, it is generally agreed that the freezing of the vital organ in vitro may be one of the most promising approaches to extending the period of preservation. The present study was designed to clarify the viability of the canine lung transplant preserved by cryogenic techniques from the histologic and functional points of view. Materials and l11ethods Cooling of the isolated lung. The left lungs of the dogs were excised and stored in a chamber -25° and -70° C. after perfusion with cold (4° C.) heparinized 5 per cent low molecular weight dextran at a pressure of 15 to 20 em. of water. The core temperature of the lung was measured automatically by a thermistor placed in the pulmonary artery, and a time-temperature curve was recorded. From the Department of Surgery, The Research Institute for Tuberculosis, Leprosy. and Cancer. Tohoku University, Sendai, Japan. Received for publication May 19, 1972. 'Director, Professor of Surgery.
180
Histologic studies were performed to evaluate the effect of cooling on the preserved lung. Reimplantation of the preserved lung.
Twenty-three adult mongrel dogs weighing 16 to 30 kilograms were used. All the animals were premedicated intravenously with 4 mg. of dexamethasone and 3 mg. per kilogram of chlorpromazine 40 minutes prior to the operation. The lung was removed from the chest and perfused with heparinized 5 per cent low molecular weight dextran containing 8 mg. per liter of dexamethasone and 100 mg. per liter of chlorpromazine at a pressure of 15 to 20 em. of water. The solution was adjusted to pH 7.4 and was precooled at 4° C. The lung was preserved at a core temperature of _3° to _5° C. for 2 to 5 hours. Then, it was warmed with water at 50° C. for 15 to 20 minutes and reimplanted. The unilateral pu1monary artery occlusion test was performed at 3, 6, and 12 weeks on the dogs that survived more than 3 weeks after reimplantation of the preserved lung. Pulmonary arterial pressure was measured continuously. Expired gas was collected in the Tissot gasometer. The oxygen uptake was determined by an expired gas analysis and the oxygen content by the method of Van Slyke and Neill. Po 2 , Pco., and pH in the arterial
Volume 65
Preservation of lung at subzero temperature
Number 2
18 1
February, 1973
°C Exdsed
30
.l,
20 10
° C Excised i 30 \ 20 Ql
~
Ql
\l-Perf~on
~ 10 20
.j.J
III l-l
10
~ 30
.j.J
~ Ql
S
0
~ 40
~ -10 Ql
E-t
50
-20 -30
o
60 \rStorClge at-25C
2
3
70
4
5
6 tnr
2
3
4
5
16 hour
Fig. 1. Freezing curve of isolated lung perfused with 5 per cent dextran precooled at 4 C. Left, Storage at -25 0 C. after perfusion. Right, Storage at -70 0 C. after perfusion. 0
blood were measured directly by the Astrup method. Pulmonary arteriography and lung scanning were also performed sequentially. The transplant was biopsied periodically, and tissue was studied by microscopic section.
Results Cooling of the isolated lung. The canine lung was placed in a chamber at -25° C and was cooled at a rate of 0.5 ° C. per minute. The core temperature of the lung reached the first plateau of -3 ° C. within 40 minutes. The temperature finally approached -25° C. after 2 hours. If the lung was cooled in the chamber at -70° c., the temperature dropped more rapidly and reached the first plateau of _5° C., where it remained for a shorter time before declining toward -70° C. (Fig. 1). Serial biopsy of the isolated lung after preservation at subzero temperatures (-25° and -70° C.) showed various degrees of dissociation of the perivascular space in both groups. Judging from the histologic changes of isolated lungs associated with these frozen-preservation techniques, the results in the first group of lungs preserved at -25 0 C. were more encouraging (Table I). Therefore, this cryogenic-storage technique
was employed in the following experiment. Reimplantation of the preserved lung. Nine lungs were preserved for 2 hours, twelve for 4 hours, and the remaining two for 5 hours. Of the 23 animals subjected to left lung autotransplantation, 4 dogs died during the operation because of failures in surgical technique. Nineteen survived more than 3 hours. Eleven of these 19 dogs were short-term survivors; 7 died from 3 hours to 8 days after operation of lung edema produced by mechanical obstruction such as thrombus, and 3 died from 6 hours to 15 days of bleeding, infection, or accident. The remaining animal was put to death 24 hours after operation. Of 8 dogs that survived more than 3 weeks, 6 survived more than 4 months and were studied sequentially up to 3 months postoperatively. Three animals are still alive and well 1 'h years after operation. The results of preserved lung reimplantation are summarized in Table II. The gross appearance of a transplanted lung preserved at the core temperature of _3° to _5° C. for 4 hours was almost normal 24 hours after the operation. Microscopic examination of this lung demonstrated mild perivascular edema and slight congestion. However, there were
The Journal of
182
Okaniwa et al.
Thoracic and Cardiovascular Surgery
Table I. Effect of cooling on isolated lung Cooling Temperature
Itemperature Organ I
Time
C./min.
-5° C. -5° c.
30 min. 6 hr.
1° C./min.
-5° C. -5° C.
30 min. 6 hr.
20° C.
7 hr.
I
-25° C. -70° C.
Rate
O.so
Room temperature
Arteriole: Bronchiole: Perivascular Desquamaedema tion
Alveolus Atrophy
I
Edema
+ + + +
+ +
++ ++ ++
+
Legend: Histologic changes in the isolated lung after various cooling conditions are designated as follows: Negative findings (-); moderate histologic changes (+); significant alterations (++).
Table II. Results of reimplantation of the preserved lung at subzero temperature Ischemic time (hr.) Survival period
Storage time (hr.)
Dog No.
2 2 2 2 2 2 2 2
106 108 109 110 111 112 113 119
4 4 4 4 4 4 4 4 4
116 120 121 122 124 126 127 129 130
6 6 6 6 5Yz 5Yz 5Yz 5Yz 5Yz
5 5
117 123
7 7
3Y2
Causes of death
1 da. 77 da. 189 da. 3 hr. 3 hr. 3 hr. 380 da. Alive
Bleeding Pneumonia Pneumonia Lung edema Lung edema Lung edema Pneumonia
1 da. 23 da. 8 da. 15 da. 6 da. Alive 6 hr. Alive 240 da.
Put to death Bleeding after left PAO test PV thrombosis Pneumonia PV thrombosis
3 da. 7 da.
Accident Pneumonia Lung edema PV thrombosis
Legend: PAO, Pulmonary artery occlusion. PV, Pulmonary vein.
no histologic findings of perivascular edema in the same lung I week after operation (Fig. 2). Pulmonary arteriograms were normal 3 weeks after operation. Small pulmonary arteries were visible on the transplanted side (Fig. 3). A pulmonary scintigram of the same dog demonstrated somewhat reduced blood flow to the transplanted side. Serial tests of unilateral pulmonary artery occlusion were performed 3 weeks, 6 weeks, and more than 12 weeks after operation. Blood gas analysis at the same time of sequential right pulmonary artery occlusion showed a consistent decrease in arterial P0 2 , which tended to be fixed at the later exam-
ination (Fig. 4). There was a consistent rise in mean pulmonary artery pressure during right pulmonary artery occlusion 3 weeks postoperatively, which remained almost two times the control level. Pulmonary blood flow showed a consistent decrease during the occlusion test. The values, which remained at a constant level during the second test, showed a definite increase during the third, except in 2 dogs in which suture line narrowing of the pulmonary artery was found (Fig. 5). Total pulmonary vascular resistance rose consistently during the first occlusion test but tended to be fixed thereafter. The min-
Volume 65 Number 2
Preservation of lung at subzero temperature
183
february , 1973
Fig. Z. Histologic appearance of the transplant which was biopsied 1 week after surgery. Histologic findings of perivascular edema are not apparent. (Hematoxylin and eosin; original magnification xI00.)
ute ventilation, which had risen during right pulmonary occlusion, tended to be decreased in later examination (Fig. 6). There were no negative correlations between total pulmonary vascular resistance and arterial P0 2 during the test (Fig. 7).
Fig. 3. Pulmonary arteriogram of dog No. 130, which was studied 3 weeks after reimplantation of the preserved lung for 2 hours at - 3 C. Small pulmonary arteries are visible on the transplanted side.
Dlscussion
mmHg
There are two methods of in vitro organ preservation: (1) suppression of the tissue metabolism and (2) active supply of oxygen and nutrients to isolated organs with maintenance of slight metabolism. In 1952 · and 1953, Blades and colleagues!' 2 reported that there were no significant morphologic changes in the isolated lung which had been ischemic for 120 minutes at normothermic temperature, but lung function was altered within 30 minutes of ischemia. Numerous experimental studies on lung preservation have been reported- 4 since the time when Blumenstock" successfully preserved the lung in vitro for 24 hours by freezing it at 4 0 C. and ventilating it with 100 ml. of air three or four times a minute. However, it is generally agreed that a safe limit for preservation of the lung in vitro is within 4 to 6 hours.v" and it is well known from studies of autotransplantation and homotransplantation in the dog that pulmonary edema in the immediate postoperative period is the major cause of failure in re-
0
100 90 80 70
60 50
2IYs ( 0 Control ~ U PAO
Storage
40
4hrs ( Jj Control A U PAO
Storage
30
.
1st
, 2nd
(3 Weeks) (6 Weeks)
3rd (12 Weeks)
Fig. 4. Serial changes in arterial oxygen tension during unilateral pulmonary artery occlusion (UPAO) test.
1 84
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Okaniwa et al.
Thorocic ond Cordiovosculor Surgery
mmHg ml/kg
40
2hrs ( 0 ta'llrol storage ,. UPAO 4hrs (AConlrol
400
Storctge AUPA 0
30
300
20 2hrs (0 Control Slorage • UPAO
10
4hrs (6 Control Storcge A UPAO
1st
(3WH-ks)
2nd
3rd
.129130:Suture line Stenosis
1st
(6Weeks) (12WeeJ
2nd
3rd
(3Weeks) (6Weeks) (12Weeks-)
Fig. 5. Serial changes in mean pulmonary artery pressure (left), and pulmonary blood flow (right) during unilateral pulmonary artery occlusion (UPAO) test.
2hrs (0 Control
Storage
ml/kg
CIt U PAO
4ITs ( l:i Contrd Storage Au PAO
800 700 600 500 400 300 200
~r,sz:::.-_0Yl9
100
1st (3Weeks)
2nd
3rd
(6Weeks) (12Weeks-)
Fig. 6. Serial changes in minute ventilation during unilateral pulmonary artery occlusion (UPAO) test.
implantation of the preserved lung if the period of ischemia is longer than 6 hours. Such a period of preservation is insufficient for the purpose of long-term preservation in vitro. It is hoped that simple effective methods of long-term storage will be developed in the future. Freezing seems to be the most promising method for longterm storage. At present, however, the freezing of organs presents a number of problems. The freezing of cells is most successful when performed relatively slowly, and the method of slow freezing is well known to be advantageous in preserving cellular functions.9 In our experiment, the core temperature of the isolated lung stored in a chamber at -25 0 C. dropped at a cooling rate of 0.5 0 C. per minute and reached the first plateau of _3 0 C. It can be considered that the formation of intracellular ice crystals does not occur at a plateau of _3 0 C. The period of the plateau may be the state of supercooling. Dimethylsulfoxide (DMSO) and glycerol are well known as the agents which protect cells from cryogenic injury. There have been studies on the preservation of organs other
Volume 65
Preservation of lung at subzero temperature
Number 2 Februory, 1973
185
nunHg
100
PaoZ 90
4 4
80e ~
oc?.0.
80
AI29
0
00
0
70
A4
°0
60
.6l30
0
"
•
" "
21Ys (0 Control
Staage
50
0 UPAO
4hrs ( A Contrd StaClge A UPAO
40 #129.130 : Suturlt line stenosis
30
4J'200
.
400
600
~oo
TPVR
1000 1200
1~
1600
SS
Dyne'sec.crtr5
Fig. 7. Correlations between total pulmonary vascular resistance (TPVRJ and arterial oxygen tension (Pa o,) during unilateral pulmonary artery occlusion (UPAO) test.
than lungs, for example, brains, hearts, and kidneys, by freezing with the use of DMSO as a protective agent. In another experiment on lung preservation, we also used DMSO as a protective agent. The lung perfused with 15 per cent DMSO was preserved at a temperature of _3° to _5° C. for 4 hours and reimplanted after warming with water at 50° C. for 20 minutes. In histologic findings of this lung, which was biopsied 1 week after operation, thickening (or proliferation) of intima and thrombi in the small vessels was observed. All the transplanted lungs perfused with the solution containing 15 to 20 per cent DMSO had the same histologic appearance (Fig. 8). In our experiment, DMSO was toxic to the endothelial cells of the pulmonary vasculature. However, it is important to point out that such protective agents as DMSO and glycerol should be very advantageous in the freezing of organs if the toxic effect of these drugs can be prevented. Lung scanning demonstrated a diminished blood flow through the reimplanted lung. This is interpreted as being due to increased resistance to the blood flow to the transplanted lung in relation to the contralateral
Fig. 8. Histologic appearance of the transplant which was preserved for 4 hours at - 50 C. following perfusion with 15 per cent dimethylsulfoxide Microscopic sections taken 7 days after reimplantation. Thickening of intima and thrombi in the small vessels is observed . (Hematoxylin and eosin; original magnification x40.)
lung. The distribution of radioactivity in the transplanted lung in which suture line narrowing of the pulmonary artery was found on pulmonary arteriogram definitely showed a diminished blood flow. The pulmonary artery pressure and the total pulmonary vascular resistance in the dog with a reimplanted, preserved lung rose consistently during the test of right pulmonary artery
The Journal of
I86
Ok aniwa et al.
Thoracic and Cordiavascular Surgery
occlusion; these values tended to be fixed 3 weeks after the operation. The minute ventilation, which rose early, during the first occlusion test, tended to be decreased in later examinations. There were no negative correlations between total pulmonary vascular resistance and arterial POz and also no significant changes in POz in the arterial blood during the test. From these results of pulmonary function, it can be considered that the reimplanted lung preserved at subzero temperatures for 2 to 5 hours functioned well during the postoperative period. While there are significant fixed hemodynamic abnormalities during the postoperative period which suggest a primary process in the pulmonary structure after preservation, these changes do not appear to constitute a serious problem that might limit longevity of dogs that have only a transplanted lung.
artery occlusion in those dogs demonstrated the functional defect of the transplant in various degrees. The defect tended to be fixed 3 weeks after the operation. These data suggest that transplanted lungs undergo a residual intrinsic change in structure. However, the cryogenic method of lung storage employed in this study still allows for the possibility of long survival with only a transplanted lung. REFERENCES
2
3
4
Summary Feasibility of cryogenic storage techniques for lung preservation was studied in dogs. These experiments included lung perfusion with cold (4°C.) heparinized 5 per cent low molecular weight dextran containing dexamethasone and chlorpromazine followed by freezing at a rate of 0.5° C. per minute. The lung was stored in a chamber at a core temperature of _3° to _5° C. for 2 to 5 hours. After rewarming, the lung was reimplanted. Six animals of 23 survived more than 4 months. Although edematous changes were observed in the preserved lung in the early postoperative period, the lung returned to normal within I week after the operation. Delayed unilateral pulmonary
5
6 7 8
9
Blades, B., Beattie, E. I., rr., Hill, R. P., and Thistlethwaite, R.: Ischemia of the Lung, Ann. Surg. 135: 56, 1952. Blades, B., Pierpont, H. C., Samadi, A., and Hill, R. P.: The Effect of Experimental Lung Ischemia on Pulmonary Function, Surg. Forum 4: 255, 1953. Largiader, F., Manax, W. G., Lyons, G. W., and Lillehei, R. C.: Technical Aspects of Transplantation of Preserved Lung, Chest 49: I, 1966. Veith, F. I., Deysine, M., Nehlson, S. L., and Karl, R. C.: Preservation of Pulmonary Function, Hemodynamics, and Morphology in Isolated Perfused Canine Lung, I. THORAC. CARDIOVASC. SURG. 52: 437, 1966. Blumenstock, D. A., Hechtman, H. B., and Collins, I. A.: Preservation of the Canine Lung, I. THORAC. CARDIOVASC. SURG. 44: 771, 1962. Hardy, I. D., Erasran, S., and Webb, W. R.: Transplantation of the Lung, Ann. Surg, 16: 440, 1964. Homatos, I., Bryant, L., and Eiseman, B.: Time Limits of Cadaver Lung 'viability, I. THORAC. CARDIOVASC. SURG. 56: 132, 1968. Ardekarni, R. G., Faber, L. R., and Beattie, E. 1., Ir.: Pulmonary Function After Various Periods of Ischemia in the Canine Lung, I. THORAC. CARDIOVASC. SURG. 59: 607, 1970. Karow, A. M., Ir., and Webb, W. R.: Principles and Problems of Hypothermic Organ Preservation, Surg. Gynecol. Obstet. 119: 609, 1964.
There have been two approaches to the problem of organ preservation; one is storage above the freezing point of water and the other is storage below the freezing point. The extensive cell damage produced by freezing and warming of large tissue masses cannot be totally avoided. However, it is generally agreed that the freezing of the vital organ in vitro may be one of the most promising approaches to extending the period of preservation. The present study was designed to clarify the viability of the canine lung transplant preserved by cryogenic techniques from the histologic and functional points of view. Materials and l11ethods Cooling of the isolated lung. The left lungs of the dogs were excised and stored in a chamber -25° and -70° C. after perfusion with cold (4° C.) heparinized 5 per cent low molecular weight dextran at a pressure of 15 to 20 em. of water. The core temperature of the lung was measured automatically by a thermistor placed in the pulmonary artery, and a time-temperature curve was recorded. From the Department of Surgery, The Research Institute for Tuberculosis, Leprosy. and Cancer. Tohoku University, Sendai, Japan. Received for publication May 19, 1972. 'Director, Professor of Surgery.
180
Histologic studies were performed to evaluate the effect of cooling on the preserved lung. Reimplantation of the preserved lung.
Twenty-three adult mongrel dogs weighing 16 to 30 kilograms were used. All the animals were premedicated intravenously with 4 mg. of dexamethasone and 3 mg. per kilogram of chlorpromazine 40 minutes prior to the operation. The lung was removed from the chest and perfused with heparinized 5 per cent low molecular weight dextran containing 8 mg. per liter of dexamethasone and 100 mg. per liter of chlorpromazine at a pressure of 15 to 20 em. of water. The solution was adjusted to pH 7.4 and was precooled at 4° C. The lung was preserved at a core temperature of _3° to _5° C. for 2 to 5 hours. Then, it was warmed with water at 50° C. for 15 to 20 minutes and reimplanted. The unilateral pu1monary artery occlusion test was performed at 3, 6, and 12 weeks on the dogs that survived more than 3 weeks after reimplantation of the preserved lung. Pulmonary arterial pressure was measured continuously. Expired gas was collected in the Tissot gasometer. The oxygen uptake was determined by an expired gas analysis and the oxygen content by the method of Van Slyke and Neill. Po 2 , Pco., and pH in the arterial
Volume 65
Preservation of lung at subzero temperature
Number 2
18 1
February, 1973
°C Exdsed
30
.l,
20 10
° C Excised i 30 \ 20 Ql
~
Ql
\l-Perf~on
~ 10 20
.j.J
III l-l
10
~ 30
.j.J
~ Ql
S
0
~ 40
~ -10 Ql
E-t
50
-20 -30
o
60 \rStorClge at-25C
2
3
70
4
5
6 tnr
2
3
4
5
16 hour
Fig. 1. Freezing curve of isolated lung perfused with 5 per cent dextran precooled at 4 C. Left, Storage at -25 0 C. after perfusion. Right, Storage at -70 0 C. after perfusion. 0
blood were measured directly by the Astrup method. Pulmonary arteriography and lung scanning were also performed sequentially. The transplant was biopsied periodically, and tissue was studied by microscopic section.
Results Cooling of the isolated lung. The canine lung was placed in a chamber at -25° C and was cooled at a rate of 0.5 ° C. per minute. The core temperature of the lung reached the first plateau of -3 ° C. within 40 minutes. The temperature finally approached -25° C. after 2 hours. If the lung was cooled in the chamber at -70° c., the temperature dropped more rapidly and reached the first plateau of _5° C., where it remained for a shorter time before declining toward -70° C. (Fig. 1). Serial biopsy of the isolated lung after preservation at subzero temperatures (-25° and -70° C.) showed various degrees of dissociation of the perivascular space in both groups. Judging from the histologic changes of isolated lungs associated with these frozen-preservation techniques, the results in the first group of lungs preserved at -25 0 C. were more encouraging (Table I). Therefore, this cryogenic-storage technique
was employed in the following experiment. Reimplantation of the preserved lung. Nine lungs were preserved for 2 hours, twelve for 4 hours, and the remaining two for 5 hours. Of the 23 animals subjected to left lung autotransplantation, 4 dogs died during the operation because of failures in surgical technique. Nineteen survived more than 3 hours. Eleven of these 19 dogs were short-term survivors; 7 died from 3 hours to 8 days after operation of lung edema produced by mechanical obstruction such as thrombus, and 3 died from 6 hours to 15 days of bleeding, infection, or accident. The remaining animal was put to death 24 hours after operation. Of 8 dogs that survived more than 3 weeks, 6 survived more than 4 months and were studied sequentially up to 3 months postoperatively. Three animals are still alive and well 1 'h years after operation. The results of preserved lung reimplantation are summarized in Table II. The gross appearance of a transplanted lung preserved at the core temperature of _3° to _5° C. for 4 hours was almost normal 24 hours after the operation. Microscopic examination of this lung demonstrated mild perivascular edema and slight congestion. However, there were
The Journal of
182
Okaniwa et al.
Thoracic and Cardiovascular Surgery
Table I. Effect of cooling on isolated lung Cooling Temperature
Itemperature Organ I
Time
C./min.
-5° C. -5° c.
30 min. 6 hr.
1° C./min.
-5° C. -5° C.
30 min. 6 hr.
20° C.
7 hr.
I
-25° C. -70° C.
Rate
O.so
Room temperature
Arteriole: Bronchiole: Perivascular Desquamaedema tion
Alveolus Atrophy
I
Edema
+ + + +
+ +
++ ++ ++
+
Legend: Histologic changes in the isolated lung after various cooling conditions are designated as follows: Negative findings (-); moderate histologic changes (+); significant alterations (++).
Table II. Results of reimplantation of the preserved lung at subzero temperature Ischemic time (hr.) Survival period
Storage time (hr.)
Dog No.
2 2 2 2 2 2 2 2
106 108 109 110 111 112 113 119
4 4 4 4 4 4 4 4 4
116 120 121 122 124 126 127 129 130
6 6 6 6 5Yz 5Yz 5Yz 5Yz 5Yz
5 5
117 123
7 7
3Y2
Causes of death
1 da. 77 da. 189 da. 3 hr. 3 hr. 3 hr. 380 da. Alive
Bleeding Pneumonia Pneumonia Lung edema Lung edema Lung edema Pneumonia
1 da. 23 da. 8 da. 15 da. 6 da. Alive 6 hr. Alive 240 da.
Put to death Bleeding after left PAO test PV thrombosis Pneumonia PV thrombosis
3 da. 7 da.
Accident Pneumonia Lung edema PV thrombosis
Legend: PAO, Pulmonary artery occlusion. PV, Pulmonary vein.
no histologic findings of perivascular edema in the same lung I week after operation (Fig. 2). Pulmonary arteriograms were normal 3 weeks after operation. Small pulmonary arteries were visible on the transplanted side (Fig. 3). A pulmonary scintigram of the same dog demonstrated somewhat reduced blood flow to the transplanted side. Serial tests of unilateral pulmonary artery occlusion were performed 3 weeks, 6 weeks, and more than 12 weeks after operation. Blood gas analysis at the same time of sequential right pulmonary artery occlusion showed a consistent decrease in arterial P0 2 , which tended to be fixed at the later exam-
ination (Fig. 4). There was a consistent rise in mean pulmonary artery pressure during right pulmonary artery occlusion 3 weeks postoperatively, which remained almost two times the control level. Pulmonary blood flow showed a consistent decrease during the occlusion test. The values, which remained at a constant level during the second test, showed a definite increase during the third, except in 2 dogs in which suture line narrowing of the pulmonary artery was found (Fig. 5). Total pulmonary vascular resistance rose consistently during the first occlusion test but tended to be fixed thereafter. The min-
Volume 65 Number 2
Preservation of lung at subzero temperature
183
february , 1973
Fig. Z. Histologic appearance of the transplant which was biopsied 1 week after surgery. Histologic findings of perivascular edema are not apparent. (Hematoxylin and eosin; original magnification xI00.)
ute ventilation, which had risen during right pulmonary occlusion, tended to be decreased in later examination (Fig. 6). There were no negative correlations between total pulmonary vascular resistance and arterial P0 2 during the test (Fig. 7).
Fig. 3. Pulmonary arteriogram of dog No. 130, which was studied 3 weeks after reimplantation of the preserved lung for 2 hours at - 3 C. Small pulmonary arteries are visible on the transplanted side.
Dlscussion
mmHg
There are two methods of in vitro organ preservation: (1) suppression of the tissue metabolism and (2) active supply of oxygen and nutrients to isolated organs with maintenance of slight metabolism. In 1952 · and 1953, Blades and colleagues!' 2 reported that there were no significant morphologic changes in the isolated lung which had been ischemic for 120 minutes at normothermic temperature, but lung function was altered within 30 minutes of ischemia. Numerous experimental studies on lung preservation have been reported- 4 since the time when Blumenstock" successfully preserved the lung in vitro for 24 hours by freezing it at 4 0 C. and ventilating it with 100 ml. of air three or four times a minute. However, it is generally agreed that a safe limit for preservation of the lung in vitro is within 4 to 6 hours.v" and it is well known from studies of autotransplantation and homotransplantation in the dog that pulmonary edema in the immediate postoperative period is the major cause of failure in re-
0
100 90 80 70
60 50
2IYs ( 0 Control ~ U PAO
Storage
40
4hrs ( Jj Control A U PAO
Storage
30
.
1st
, 2nd
(3 Weeks) (6 Weeks)
3rd (12 Weeks)
Fig. 4. Serial changes in arterial oxygen tension during unilateral pulmonary artery occlusion (UPAO) test.
1 84
The Journol of
Okaniwa et al.
Thorocic ond Cordiovosculor Surgery
mmHg ml/kg
40
2hrs ( 0 ta'llrol storage ,. UPAO 4hrs (AConlrol
400
Storctge AUPA 0
30
300
20 2hrs (0 Control Slorage • UPAO
10
4hrs (6 Control Storcge A UPAO
1st
(3WH-ks)
2nd
3rd
.129130:Suture line Stenosis
1st
(6Weeks) (12WeeJ
2nd
3rd
(3Weeks) (6Weeks) (12Weeks-)
Fig. 5. Serial changes in mean pulmonary artery pressure (left), and pulmonary blood flow (right) during unilateral pulmonary artery occlusion (UPAO) test.
2hrs (0 Control
Storage
ml/kg
CIt U PAO
4ITs ( l:i Contrd Storage Au PAO
800 700 600 500 400 300 200
~r,sz:::.-_0Yl9
100
1st (3Weeks)
2nd
3rd
(6Weeks) (12Weeks-)
Fig. 6. Serial changes in minute ventilation during unilateral pulmonary artery occlusion (UPAO) test.
implantation of the preserved lung if the period of ischemia is longer than 6 hours. Such a period of preservation is insufficient for the purpose of long-term preservation in vitro. It is hoped that simple effective methods of long-term storage will be developed in the future. Freezing seems to be the most promising method for longterm storage. At present, however, the freezing of organs presents a number of problems. The freezing of cells is most successful when performed relatively slowly, and the method of slow freezing is well known to be advantageous in preserving cellular functions.9 In our experiment, the core temperature of the isolated lung stored in a chamber at -25 0 C. dropped at a cooling rate of 0.5 0 C. per minute and reached the first plateau of _3 0 C. It can be considered that the formation of intracellular ice crystals does not occur at a plateau of _3 0 C. The period of the plateau may be the state of supercooling. Dimethylsulfoxide (DMSO) and glycerol are well known as the agents which protect cells from cryogenic injury. There have been studies on the preservation of organs other
Volume 65
Preservation of lung at subzero temperature
Number 2 Februory, 1973
185
nunHg
100
PaoZ 90
4 4
80e ~
oc?.0.
80
AI29
0
00
0
70
A4
°0
60
.6l30
0
"
•
" "
21Ys (0 Control
Staage
50
0 UPAO
4hrs ( A Contrd StaClge A UPAO
40 #129.130 : Suturlt line stenosis
30
4J'200
.
400
600
~oo
TPVR
1000 1200
1~
1600
SS
Dyne'sec.crtr5
Fig. 7. Correlations between total pulmonary vascular resistance (TPVRJ and arterial oxygen tension (Pa o,) during unilateral pulmonary artery occlusion (UPAO) test.
than lungs, for example, brains, hearts, and kidneys, by freezing with the use of DMSO as a protective agent. In another experiment on lung preservation, we also used DMSO as a protective agent. The lung perfused with 15 per cent DMSO was preserved at a temperature of _3° to _5° C. for 4 hours and reimplanted after warming with water at 50° C. for 20 minutes. In histologic findings of this lung, which was biopsied 1 week after operation, thickening (or proliferation) of intima and thrombi in the small vessels was observed. All the transplanted lungs perfused with the solution containing 15 to 20 per cent DMSO had the same histologic appearance (Fig. 8). In our experiment, DMSO was toxic to the endothelial cells of the pulmonary vasculature. However, it is important to point out that such protective agents as DMSO and glycerol should be very advantageous in the freezing of organs if the toxic effect of these drugs can be prevented. Lung scanning demonstrated a diminished blood flow through the reimplanted lung. This is interpreted as being due to increased resistance to the blood flow to the transplanted lung in relation to the contralateral
Fig. 8. Histologic appearance of the transplant which was preserved for 4 hours at - 50 C. following perfusion with 15 per cent dimethylsulfoxide Microscopic sections taken 7 days after reimplantation. Thickening of intima and thrombi in the small vessels is observed . (Hematoxylin and eosin; original magnification x40.)
lung. The distribution of radioactivity in the transplanted lung in which suture line narrowing of the pulmonary artery was found on pulmonary arteriogram definitely showed a diminished blood flow. The pulmonary artery pressure and the total pulmonary vascular resistance in the dog with a reimplanted, preserved lung rose consistently during the test of right pulmonary artery
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occlusion; these values tended to be fixed 3 weeks after the operation. The minute ventilation, which rose early, during the first occlusion test, tended to be decreased in later examinations. There were no negative correlations between total pulmonary vascular resistance and arterial POz and also no significant changes in POz in the arterial blood during the test. From these results of pulmonary function, it can be considered that the reimplanted lung preserved at subzero temperatures for 2 to 5 hours functioned well during the postoperative period. While there are significant fixed hemodynamic abnormalities during the postoperative period which suggest a primary process in the pulmonary structure after preservation, these changes do not appear to constitute a serious problem that might limit longevity of dogs that have only a transplanted lung.
artery occlusion in those dogs demonstrated the functional defect of the transplant in various degrees. The defect tended to be fixed 3 weeks after the operation. These data suggest that transplanted lungs undergo a residual intrinsic change in structure. However, the cryogenic method of lung storage employed in this study still allows for the possibility of long survival with only a transplanted lung. REFERENCES
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Summary Feasibility of cryogenic storage techniques for lung preservation was studied in dogs. These experiments included lung perfusion with cold (4°C.) heparinized 5 per cent low molecular weight dextran containing dexamethasone and chlorpromazine followed by freezing at a rate of 0.5° C. per minute. The lung was stored in a chamber at a core temperature of _3° to _5° C. for 2 to 5 hours. After rewarming, the lung was reimplanted. Six animals of 23 survived more than 4 months. Although edematous changes were observed in the preserved lung in the early postoperative period, the lung returned to normal within I week after the operation. Delayed unilateral pulmonary
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Blades, B., Beattie, E. I., rr., Hill, R. P., and Thistlethwaite, R.: Ischemia of the Lung, Ann. Surg. 135: 56, 1952. Blades, B., Pierpont, H. C., Samadi, A., and Hill, R. P.: The Effect of Experimental Lung Ischemia on Pulmonary Function, Surg. Forum 4: 255, 1953. Largiader, F., Manax, W. G., Lyons, G. W., and Lillehei, R. C.: Technical Aspects of Transplantation of Preserved Lung, Chest 49: I, 1966. Veith, F. I., Deysine, M., Nehlson, S. L., and Karl, R. C.: Preservation of Pulmonary Function, Hemodynamics, and Morphology in Isolated Perfused Canine Lung, I. THORAC. CARDIOVASC. SURG. 52: 437, 1966. Blumenstock, D. A., Hechtman, H. B., and Collins, I. A.: Preservation of the Canine Lung, I. THORAC. CARDIOVASC. SURG. 44: 771, 1962. Hardy, I. D., Erasran, S., and Webb, W. R.: Transplantation of the Lung, Ann. Surg, 16: 440, 1964. Homatos, I., Bryant, L., and Eiseman, B.: Time Limits of Cadaver Lung 'viability, I. THORAC. CARDIOVASC. SURG. 56: 132, 1968. Ardekarni, R. G., Faber, L. R., and Beattie, E. 1., Ir.: Pulmonary Function After Various Periods of Ischemia in the Canine Lung, I. THORAC. CARDIOVASC. SURG. 59: 607, 1970. Karow, A. M., Ir., and Webb, W. R.: Principles and Problems of Hypothermic Organ Preservation, Surg. Gynecol. Obstet. 119: 609, 1964.