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Lung transplantation
Lung transplantation Hypothermic storage for 24 hours in a colloid hyperosmolar solution We studied the effects of a colloid hyperosmolar solution in preservation of lungs for transplantation in dogs. Fresh allografts were compared to lungs stored (4 to 7° C.) in Ringer's lactate for 3 hours and in modified silica gel fraction (MSGF) for 8, 16, and 24 hours before transplantation. Lungs preserved in Ringer's lactate for 3 hours were significantly damaged, and there were no long-term survivors in recipient dogs (1.2 ± 0.4 days) (mean ± S.E.). In contrast, the recipients of lungs preserved in MSGF for periods of up to 24 hours lived as long as the recipients of fresh allografts flushed with Ringer's lactate or MSGF (24 hour stored MSGF grafts = 10.5 ± 3.1 days, survival, mean ± S.E.; fresh Ringer's lactate grafts = 8.5 ± 2.3 days, mean ± S.E.; fresh MSGF grafts =13.6 ± 3.8 days, mean ± S.E.). Arterial blood gas measurements and chest roentgenograms were good methods of assessing the condition of preserved lung allografts. No significant differences were observed between the fresh and MSGF-preserved grafts. Pneumonia and rejection were the most frequent causes of death for both the fresh and MSGF-preserved allograft recipients. We demonstrate that a colloid hyperosmolar solution (MSGF) is a good method for 24 hour hypothermic storage of lung allografts for transplantation.
Luis H. Toledo-Pereyra, M . D . , Ph.D., Detroit, Mich., and Richard M. Condie, Minneapolis, Minn.
J.V.Lany attempts have been made to preserve the lungs under hypothermic conditions for transplantation. 1 - 1 3 Hypothermic pulsatile perfusion of canine lungs for 72 hours has been studied ex vivo. 9 Veith and his group 13 have also shown hypothermic storage with Sacks solution to be effective. Recently, ToledoPereyra and his associates 11 showed the beneficial effects of a colloid hyperosmolar solution for 72 hours of hypothermic storage of lungs before transplantation. However, the contralateral pulmonary vessels were ligated 3 days after transplantation. In this article, we report on the effect of the same colloid hyperosmolar From the Department of Surgery, Section of Transplantation and Surgical Research, Henry Ford Hospital, Detroit, Mich., and the Department of Surgery, University of Minnesota, Minneapolis, Minn. This work was partially supported by Henry Ford Hospital Institutional Grant No. 730-0786 from the Ford Foundation. Received for publication April 5, 1978. Accepted for publication Aug. 14, 1978. Address for reprints: Dr. Luis H. Toledo-Pereyra, Department of Surgery, Section of Transplantation and Surgical Research, 2799 W. Grand Blvd., Detroit, Mich. 48202. 846
solution 11 used for 24 hours' hypothermic storage of lung allografts transplanted into recipients, followed by immediate ligation of the contralateral pulmonary artery. Material and methods Experimental design. Adult mongrel dogs of both sexes, weighing between 17 to 24 kilograms, were anesthetized, with sodium methohexital for induction and halothane for maintenance (0.5 to 2 percent) of anesthesia. One gram of methylprednisolone was administered intravenously before the dissection was begun. The donor dogs underwent a left thoracotomy, the chest was entered at the fourth intercostal space, the pulmonary hilus was identified, and the vessels and the main bronchus were isolated. The hilar structures were divided 10 minutes after administering 2,000 U. of heparin intravenously. The lung was then flushed with 300 to 500 ml. of Ringer's lactate or modified silica gel fraction (MSGF), which is basically a plasma fraction treated with silica gel, resulting in removal of all cholesterol and fibrinogen and most lipoproteins. 8 MSGF was made hyperosmolar (420 to 450 mOsm. per
0022-5223/78/ 120846+07S00.70/0 © 1978 The C. V. Mosby Co.
Volume 76 Number 6 December, 1978
Lung transplantation
847
I RL or SGF or MSGF
DOG A (DONOR)
8-24 hrs 4-7° C
LUNG REMOVAL
LUNG TRANSPLANT
I gm Methylprednisolone 2000 U Heparin
Azathioprine 5.0 mg/K/day x 3 days 2.5 mg/ K/day until death
Clinical Laboratory Assessment Blood Counts Blood Gases Chest X-ray Histology D 0 G B (RECIPIENT)
Immediate contralateral pulmonary artery ligation
Fig. 1. Experimental design to test the effect of a new colloid hyperosmolar solution (MSGF, modified silica gel fraction). Hypothermic storage of lung allografts for 24 hours, transplantion and then immediate contralateral pulmonary artery ligation in recipients. RL, Ringer's lactate. SGF, Silica gel fraction. Table I. Characteristics of all groups of stored and unstored transplanted lungs Groups
No of dogs
Storage solution
Duration of storage at 4-7 >C.(hr.)
I II
8 8 8 8 8 8
Ringer's lactate Ringer's lactate MSGF MSGF MSGF MSGF
0 3 0 8 16 24
in
IV V VI
Survival in days (Mean ± S.E.) 8.5 1.2 13.6 9.8 11.2 10.5
± ± ± ± ± ±
2.3 0.4* 3.8 2.7 2.7 3.1
Longest survival time (days) 15 2 47 25 29 34
*p < 0.005 when compared to Groups I, II, IV, V, and VI.
liter) with albumin and glucose, and hyperkalemic (100 mEq. per liter) with potassium. The final concentration of albumin was 4.1 to 5.0 Gm. per liter; total proteins, 6.1 to 6.8 Gm. per liter; and glucose, 1 Gm. per liter. Additives to this solution included methylprednisolone (500 mg. per liter) and insulin (80 U. per liter). The flushing conditons were hypothermia at 15° C. and a height of 40 cm. The flushing solution was maintained within the lung by clamping the vascular structures during the entire storage period. The flushing time, the time needed to flush 300 to 500 ml of solution, averaged 12 minutes. The lungs were partially inflated (20 to 40 percent) with 99.96 percent nitrogen* *Liquid Carbonics, Chicago, 111.
administered slowly through the main bronchus. Thereafter, the bronchus was closed with an atraumatic clamp for the preservation period. The lungs were partially immersed in the same solution utilized for the initial flushing and were preserved under hypothermic storage conditons (4 to 7° C.) for variable periods of time (Table I). The lungs were weighed before and at the end of storage. After hypothermic storage (3 to 24 hours) or immediately after flushing, the left lung was transplanted into a recipient after excision of its left lung, leaving lung vascular cuffs for the anastomoses as well as for the main bronchus. The vascular anastomoses were performed with 5-0 and 6-0 Prolene for the left atrial cuff and pulmonary artery, respectively. Special atten-
The Journal of Thoracic and Cardiovascular Surgery
8 4 8 Toledo-Pereyra and Condie
Table II. Cause of death of recipients of all fresh and preserved lung allografts
100 Fresh transplants ■o
Groups
o
Cause of death
0
1
2
3 Days
4
5
6
II
III
IV
VI
Hemorrhagic lung Pulmonary edema Pneumonia Rejection Pneumonia and rejection Unknown
0 0 2 2 3 1
4 3 1 0 0 0
0 0 2 2 3 1
0 0 2 1 3 2
0 0 1 2 4 1
0 1 2 3 2 0
Totals
8
8
8
8
8
8
Posttransplantation
Fig. 2. Average postoperative values of arterial oxygen tension in dogs that received fresh allografts or in animals that received lungs that had been stored for 24 hours (7° C.) in Ringer's solution or MSGF. tion was given to the pulmonary artery, where very small bites were taken to avoid any lack of distensibility or future technical problems. The bronchus was anastomosed by the usual method, as described by Veith and Richards.14 As soon as the transplant operation was finished, the right contralateral pulmonary artery was ligated,15 and the chest was closed in the usual fashion. In the recipient minimal immunosuppression was provided with azathioprine, 5.0 mg. per kilogram of body weight per day for 3 days and then 2.5 mg. per kilogram per day until death (Fig. 1). Laboratory studies. Immediately after operation we began measuring blood gases every hour for 4 hours, and daily thereafter. All animals also underwent daily chest x-ray and complete blood counts for 1 week. Thereafter, biweekly chest x-ray films and complete blood counts were taken until death. Postmortem examination was performed in all dogs by analyzing the transplanted lung as well as the contralateral side that had been ligated. All lung samples were fixed in 10 percent formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined under the light microscope. Experimental variables. Six groups of dogs were prepared, with eight dogs provided in each study group. Group I received fresh lungs flushed with Ringer's lactate and transplanted immediately; in Group II the lungs were flushed with Ringer's solution, stored for 3 hours at 4 to 7 ° C , and then transplanted; in Group III the lungs were flushed with MSGF and transplanted immediately; Groups IV, V, and VI received lungs that had been flushed with MSGF and stored under hypothermic conditions (4 to 7° C.) for 8,
16, and 24 hours, respectively. If a dog died as a result of technical failure a substitute dog was prepared for that group to maintain a total of eight dogs each. Of the five dogs that died because of technical failure, one died of pulmonary artery bleeding (Group I); one died with severe atrial cuff obstruction (Group II); one died with a significant bronchial stump leak (Group IV); another developed pulmonary edema and respiratory insufficiency (Group V); and the last died of anesthesia complications (Group II). Table II includes only the dogs in which there were no technical failures. Student's t test was utilized to determine all statistical variables in this group of transplanted lungs. Results Table I shows the average survival times of all groups of dogs. It was evident that hypothermic storage with a colloid hyperosmolar solution protected the lungs for 24 hours before transplantation. Recipients of lungs stored with this solution lived as long as the recipients of fresh allografts. In contrast, lungs stored in Ringer's lactate for only 3 hours were significantly damaged after transplantation, and there were no 3 day survivors. Upon transplantation, the recipients of fresh lungs as well as those with lungs stored in MSGF solution had good arterial blood gas values (Fig. 2). The Po2 remained above 70 mm. Hg with no evidence of respiratory failure. The recipients of lungs stored in Ringer's lactate for 3 hours had severe drop in Po 2 immediately after transplantation (Fig. 2). The Po2 deteriorated before death in all animals. In all animals that did not experience respiratory distress, the arterial Pco2 and pH were normal. The most sensitive functional index in these transplant animals was the arterial Po 2 , as Veith and his associates15 suggested previously. Radiologic assessment. There were no significant
Volume 76 Number 6 December, 1978
Lung transplantation
849
Fig. 3. Frontal (A) and lateral (B) plain chest roentgenograms of a dog 5 days after receiving a fresh lung transplant on the left side followed by ligation of the contralateral pulmonary artery. differences upon radiologic evaluation between lungs preserved with the colloid hyperosmolar solution and the fresh lung allografts. Minimal radiologic infiltrates were observed within the first week after transplantation of the MSGF-stored group of lungs and the fresh allografts (Figs. 3 and 4). Recipients of lungs stored in Ringer's solution for 3 hours did not live long enough to permit comparative assessment of the radiologic response after transplantation. Cause of death. Table II shows the cause of death in all groups of recipients of fresh and preserved lung allografts. Pneumonia and rejection were common causes of death in the group of animals that survived for more than 7 days after transplantation. It is also worth noting, as Veith and colleagues14 have previously reported, that pneumonia occurred as frequently in the right lung as in the left lung. Pulmonary hemorrhage and edema were present in several animals that died in the immediate post-transplantation period. Histologic findings. Fresh lung allografts showed normal structure after transplantation. Lungs stored under hypothermic conditions in Ringer's solution for 3 hours showed significant alterations immediately after transplantation, such as interstitial edema, hemorrhagic necrosis, and disrupture of the alveolocapillary barrier. Some of the lungs stored in MSGF for periods of up to
24 hours were histologically normal immediately after transplantation (Fig. 5). Discussion Our results confirm the findings of others that it is possible to preserve lung allografts under hypothermic conditions for 24 hours.6, 13 Our study also indicates that a colloid hyperosmolar solution (MSGF) is a good medium for 24 hour lung preservation. There were no significant functional or structural differences between the fresh allografts and the lungs stored under hypothermic conditions in MSGF for up to 24 hours. Many methods of lung preservation have been used in the past. 1-13 Although it has proved effective for up to 72 hours in vitro,9 hypothermic pulsatile perfusion has not been tested in vivo. Other methods, such as hyperbaric oxygenation, have not been proved to be satisfactory in part because testing has not been adequate. Recently, one group6' 13 demonstrated that a hyperosmolar, intracellular crystalloid solution (Sacks solution) is a good medium for preservation of lung allografts under hypothermic conditions for up to 24 hours. When recipients of preserved lung allografts underwent immediate contralateral pulmonary artery ligation, no abnormal changes were observed in these allografts as
8 5 0 Toledo-Pereyra and Condie
The Journal of Thoracic and Cardiovascular Surgery
Fig. 4. Frontal (A) and lateral (B) plain chest roentgenograms of a dog 5 days after receiving a transplant of a lung preserved for 24 hours in MSGF (7° C ) . The right contralateral pulmonary artery has been ligated. There were no significant abnormalities seen in the lungs, except for a moderate infiltrate in the midfjeld of the right lung.
Fig. 5. Photomicrograph of a section of lung preserved fpr 24 hours in MSGF. This sample was taken 4 days after transplantation. Morphology is normal. (Hematoxylin and eosin. Original magnification (x375.)
Volume 76 Number 6 December, 1978
compared to control fresh allografts. It was not determined whether the important factors were related to hyperosmolarity, intracellular composition, or pH. Our experimental model in this study was the canine lung allograft, as in the studies by Veith and his associates. 13-16 Therefore, rejection and immunosuppression are limiting factors in testing the adequate functional response of the transplanted lung. The use of an autotransplant would obviously eliminate these factors. However, the difficulties involved in maintaining life in a pneumonectomized dog for long periods of time outweigh the potential advantages. Furthermore, technical factors, such as diminished length of pulmonary artery and left atrium suitable for anastomosis, make the use of the lung autograft model impractical for preservation studies. In any event, it is of considerable importance in lung preservation work to test fully the functional adequacy of the preserved lungs, as done by Kondo and associates5 with bilateral lung transplants or as done in the present work and a previous study by Veith's group13 with ligation of the opposite pulmonary artery. The exact mechanism for the beneficial effect of the colloid hyperosmolar solution is not known. It is possible that the hyperosmolarity, the high concentration of proteins, the high concentration of glucose, and the low concentrations of lipids or aggregable material play a significant role in extending long-term function of preserved allografts. On the other hand, it is worth noting that Veith and co-workers13 obtained similar results with a crystalloid, hyperosmolar intracellular solution. The common characteristics of both solutions, however, are attributable to different factors. In the MSGF solution, the albumin, glucose, and potassium contributed to the high osmolarity, while in the Sacks solution the hyperosmolarity was due to mannitol. Taking these findings into consideration, hyperosmolarity and hyperkalemia appear to be the most consistent factors in maintaining the viability of preserved lung allografts. Further studies in this area should clarify some of these theoretical implications. Although we have found that preservation at low temperatures (4 to 7° C.) is important, we do not think that hypothermia is, the most important fagtor in al{ cases, since 3 hours of hyppthermic preservation with a crystalloid extracellular type of solutjpn (Ringer's lactate) caused significant damage to preserved lung allografts. Hence, other factors besides hypothermia are also important. These factors are probably associated with the type of solution utilized during the period of storage, intracellular composition and hyperosmolarity
Lung transplantation
85 1
being of utmost importance, as has been mentioned previously. In summary, these results confirm that lung preservation can be achieved for 24 hours with a colloid hyperosmolar solution (MSGF). A good response was observed after transplantation and immediate contralateral pulmonary artery ligation in recipients. REFERENCES 1 Blumenstock DA, Hechtman HB, Collins JA: Preservation of the canine lung. J THORAC CARDIOVASC SURG 44:771-775, 1962 2 Blumenstock DA, Lempert N, Morgado F: Preservation of the canine lung in vitro for 24 hours with the use of hypothermia and hyperbaric oxygen. J THORAC CARDIOVASC SURG 50:769-774, 1965 3 Castagna JT, Shors E, Benfield JR: The role of perfusion in lung preservation. J THORAC CARDIOVASC SURG 63:521-526, 1972 4 Connaughton PJ, Bahuth JJ, Lewis FJ: Lung ischemia up to six hours. Influence of local cooling in situ on subsequent pulmonary function. Chest 41:404-408, 1962 5 Kondo Y, Turner D, Cokrell JV, Hardy JD: Ischemic tolerance of the canine autotransplanted lung. Surgery 76:447-453, 1974 6 Merav AD, Crane R, Pinsker KL, Novin AJ, Koerner SK, Veith FJ: Preservation, transportation, and transplantation of lungs obtained after death. Surg Forum 28:195197, 1977 7 Stevens GH, Sanchez MN, Chappell G, Bennett LR, Gyepes MT, Fonkalsrud EW: Prolonged lung allograft preservation using inbred beagles. J Surg Res 12:246253, 1972 8 Toledo-Pereyra LH, Condie RM, Malmberg R, Simmons RL, Najarian JS: Long-term kidney preservation with a new plasma perfusate. Proc Clin Dial Transplant Forum 3:88-90, 1973 9 Toledo-Pereyra LH, Northrup WF, Humphrey ED, Najarian JS: Maintenance of lung viability for transplantation after long periods of hypothermic perfusion. J Surg Res 18:99-106, 1975 10 Toledo-Pereyra LH, Hau T, Simmons RL, Najarian JS: Lung preservation techniques. Ann Thorac Surg 23:487494, 1977 11 Toledo-Pergyra LH, Condie RM, Hau J, Simmons RL, Najarian JS; Three-day hyppthermic storage pf canine lungs. Surg Forum 28:19,4-19/7, 1977 12 Toledo-Pereyra LH, Simmons RL, Najarian JS: Factors determining successful liver preservation for transplantation. Ann Surg 181:289-298, 1975 13 Veith FJ, Crane R, Torres M, Colon I, Hagstrom JW, Pinsker K, Koerner SK: Effective preservation and transportation of lung transplants. J THORAC CARDIOVASC SURG 72:97-105, 1976
8 5 2 Toledo-Pereyra and Condie
14 VeithFJ, Richards K: Improved technique for canine lung transplantation. Ann Surg 171:553-558, 1970 15 Veith FJ, Sinha SBP, Siegelman SS, Hagstrom JWC: Single lung transplantation with immediate ligation of the opposite pulmonary artery in the dog. A model for assessing the functional adequacy of transplanted lungs, Research Animals in Medicine, Lt Harmison, ed., Bethesda,
The Journal of Thoracic and Cardiovascular Surgery
Md., 1973, Department of Health, Education, and Welfare Publication No. 72-333, pp 437-445 16 Veith FJ, Sinha SBP, Graves JS, Boley SJ, Daugherty JC: Ischemic tolerance of the lung. The effect of ventilation and inflation. J THORAC CARDIOVASC SURG 61:804810, 1971
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Luis H. Toledo-Pereyra, M . D . , Ph.D., Detroit, Mich., and Richard M. Condie, Minneapolis, Minn.
J.V.Lany attempts have been made to preserve the lungs under hypothermic conditions for transplantation. 1 - 1 3 Hypothermic pulsatile perfusion of canine lungs for 72 hours has been studied ex vivo. 9 Veith and his group 13 have also shown hypothermic storage with Sacks solution to be effective. Recently, ToledoPereyra and his associates 11 showed the beneficial effects of a colloid hyperosmolar solution for 72 hours of hypothermic storage of lungs before transplantation. However, the contralateral pulmonary vessels were ligated 3 days after transplantation. In this article, we report on the effect of the same colloid hyperosmolar From the Department of Surgery, Section of Transplantation and Surgical Research, Henry Ford Hospital, Detroit, Mich., and the Department of Surgery, University of Minnesota, Minneapolis, Minn. This work was partially supported by Henry Ford Hospital Institutional Grant No. 730-0786 from the Ford Foundation. Received for publication April 5, 1978. Accepted for publication Aug. 14, 1978. Address for reprints: Dr. Luis H. Toledo-Pereyra, Department of Surgery, Section of Transplantation and Surgical Research, 2799 W. Grand Blvd., Detroit, Mich. 48202. 846
solution 11 used for 24 hours' hypothermic storage of lung allografts transplanted into recipients, followed by immediate ligation of the contralateral pulmonary artery. Material and methods Experimental design. Adult mongrel dogs of both sexes, weighing between 17 to 24 kilograms, were anesthetized, with sodium methohexital for induction and halothane for maintenance (0.5 to 2 percent) of anesthesia. One gram of methylprednisolone was administered intravenously before the dissection was begun. The donor dogs underwent a left thoracotomy, the chest was entered at the fourth intercostal space, the pulmonary hilus was identified, and the vessels and the main bronchus were isolated. The hilar structures were divided 10 minutes after administering 2,000 U. of heparin intravenously. The lung was then flushed with 300 to 500 ml. of Ringer's lactate or modified silica gel fraction (MSGF), which is basically a plasma fraction treated with silica gel, resulting in removal of all cholesterol and fibrinogen and most lipoproteins. 8 MSGF was made hyperosmolar (420 to 450 mOsm. per
0022-5223/78/ 120846+07S00.70/0 © 1978 The C. V. Mosby Co.
Volume 76 Number 6 December, 1978
Lung transplantation
847
I RL or SGF or MSGF
DOG A (DONOR)
8-24 hrs 4-7° C
LUNG REMOVAL
LUNG TRANSPLANT
I gm Methylprednisolone 2000 U Heparin
Azathioprine 5.0 mg/K/day x 3 days 2.5 mg/ K/day until death
Clinical Laboratory Assessment Blood Counts Blood Gases Chest X-ray Histology D 0 G B (RECIPIENT)
Immediate contralateral pulmonary artery ligation
Fig. 1. Experimental design to test the effect of a new colloid hyperosmolar solution (MSGF, modified silica gel fraction). Hypothermic storage of lung allografts for 24 hours, transplantion and then immediate contralateral pulmonary artery ligation in recipients. RL, Ringer's lactate. SGF, Silica gel fraction. Table I. Characteristics of all groups of stored and unstored transplanted lungs Groups
No of dogs
Storage solution
Duration of storage at 4-7 >C.(hr.)
I II
8 8 8 8 8 8
Ringer's lactate Ringer's lactate MSGF MSGF MSGF MSGF
0 3 0 8 16 24
in
IV V VI
Survival in days (Mean ± S.E.) 8.5 1.2 13.6 9.8 11.2 10.5
± ± ± ± ± ±
2.3 0.4* 3.8 2.7 2.7 3.1
Longest survival time (days) 15 2 47 25 29 34
*p < 0.005 when compared to Groups I, II, IV, V, and VI.
liter) with albumin and glucose, and hyperkalemic (100 mEq. per liter) with potassium. The final concentration of albumin was 4.1 to 5.0 Gm. per liter; total proteins, 6.1 to 6.8 Gm. per liter; and glucose, 1 Gm. per liter. Additives to this solution included methylprednisolone (500 mg. per liter) and insulin (80 U. per liter). The flushing conditons were hypothermia at 15° C. and a height of 40 cm. The flushing solution was maintained within the lung by clamping the vascular structures during the entire storage period. The flushing time, the time needed to flush 300 to 500 ml of solution, averaged 12 minutes. The lungs were partially inflated (20 to 40 percent) with 99.96 percent nitrogen* *Liquid Carbonics, Chicago, 111.
administered slowly through the main bronchus. Thereafter, the bronchus was closed with an atraumatic clamp for the preservation period. The lungs were partially immersed in the same solution utilized for the initial flushing and were preserved under hypothermic storage conditons (4 to 7° C.) for variable periods of time (Table I). The lungs were weighed before and at the end of storage. After hypothermic storage (3 to 24 hours) or immediately after flushing, the left lung was transplanted into a recipient after excision of its left lung, leaving lung vascular cuffs for the anastomoses as well as for the main bronchus. The vascular anastomoses were performed with 5-0 and 6-0 Prolene for the left atrial cuff and pulmonary artery, respectively. Special atten-
The Journal of Thoracic and Cardiovascular Surgery
8 4 8 Toledo-Pereyra and Condie
Table II. Cause of death of recipients of all fresh and preserved lung allografts
100 Fresh transplants ■o
Groups
o
Cause of death
0
1
2
3 Days
4
5
6
II
III
IV
VI
Hemorrhagic lung Pulmonary edema Pneumonia Rejection Pneumonia and rejection Unknown
0 0 2 2 3 1
4 3 1 0 0 0
0 0 2 2 3 1
0 0 2 1 3 2
0 0 1 2 4 1
0 1 2 3 2 0
Totals
8
8
8
8
8
8
Posttransplantation
Fig. 2. Average postoperative values of arterial oxygen tension in dogs that received fresh allografts or in animals that received lungs that had been stored for 24 hours (7° C.) in Ringer's solution or MSGF. tion was given to the pulmonary artery, where very small bites were taken to avoid any lack of distensibility or future technical problems. The bronchus was anastomosed by the usual method, as described by Veith and Richards.14 As soon as the transplant operation was finished, the right contralateral pulmonary artery was ligated,15 and the chest was closed in the usual fashion. In the recipient minimal immunosuppression was provided with azathioprine, 5.0 mg. per kilogram of body weight per day for 3 days and then 2.5 mg. per kilogram per day until death (Fig. 1). Laboratory studies. Immediately after operation we began measuring blood gases every hour for 4 hours, and daily thereafter. All animals also underwent daily chest x-ray and complete blood counts for 1 week. Thereafter, biweekly chest x-ray films and complete blood counts were taken until death. Postmortem examination was performed in all dogs by analyzing the transplanted lung as well as the contralateral side that had been ligated. All lung samples were fixed in 10 percent formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined under the light microscope. Experimental variables. Six groups of dogs were prepared, with eight dogs provided in each study group. Group I received fresh lungs flushed with Ringer's lactate and transplanted immediately; in Group II the lungs were flushed with Ringer's solution, stored for 3 hours at 4 to 7 ° C , and then transplanted; in Group III the lungs were flushed with MSGF and transplanted immediately; Groups IV, V, and VI received lungs that had been flushed with MSGF and stored under hypothermic conditions (4 to 7° C.) for 8,
16, and 24 hours, respectively. If a dog died as a result of technical failure a substitute dog was prepared for that group to maintain a total of eight dogs each. Of the five dogs that died because of technical failure, one died of pulmonary artery bleeding (Group I); one died with severe atrial cuff obstruction (Group II); one died with a significant bronchial stump leak (Group IV); another developed pulmonary edema and respiratory insufficiency (Group V); and the last died of anesthesia complications (Group II). Table II includes only the dogs in which there were no technical failures. Student's t test was utilized to determine all statistical variables in this group of transplanted lungs. Results Table I shows the average survival times of all groups of dogs. It was evident that hypothermic storage with a colloid hyperosmolar solution protected the lungs for 24 hours before transplantation. Recipients of lungs stored with this solution lived as long as the recipients of fresh allografts. In contrast, lungs stored in Ringer's lactate for only 3 hours were significantly damaged after transplantation, and there were no 3 day survivors. Upon transplantation, the recipients of fresh lungs as well as those with lungs stored in MSGF solution had good arterial blood gas values (Fig. 2). The Po2 remained above 70 mm. Hg with no evidence of respiratory failure. The recipients of lungs stored in Ringer's lactate for 3 hours had severe drop in Po 2 immediately after transplantation (Fig. 2). The Po2 deteriorated before death in all animals. In all animals that did not experience respiratory distress, the arterial Pco2 and pH were normal. The most sensitive functional index in these transplant animals was the arterial Po 2 , as Veith and his associates15 suggested previously. Radiologic assessment. There were no significant
Volume 76 Number 6 December, 1978
Lung transplantation
849
Fig. 3. Frontal (A) and lateral (B) plain chest roentgenograms of a dog 5 days after receiving a fresh lung transplant on the left side followed by ligation of the contralateral pulmonary artery. differences upon radiologic evaluation between lungs preserved with the colloid hyperosmolar solution and the fresh lung allografts. Minimal radiologic infiltrates were observed within the first week after transplantation of the MSGF-stored group of lungs and the fresh allografts (Figs. 3 and 4). Recipients of lungs stored in Ringer's solution for 3 hours did not live long enough to permit comparative assessment of the radiologic response after transplantation. Cause of death. Table II shows the cause of death in all groups of recipients of fresh and preserved lung allografts. Pneumonia and rejection were common causes of death in the group of animals that survived for more than 7 days after transplantation. It is also worth noting, as Veith and colleagues14 have previously reported, that pneumonia occurred as frequently in the right lung as in the left lung. Pulmonary hemorrhage and edema were present in several animals that died in the immediate post-transplantation period. Histologic findings. Fresh lung allografts showed normal structure after transplantation. Lungs stored under hypothermic conditions in Ringer's solution for 3 hours showed significant alterations immediately after transplantation, such as interstitial edema, hemorrhagic necrosis, and disrupture of the alveolocapillary barrier. Some of the lungs stored in MSGF for periods of up to
24 hours were histologically normal immediately after transplantation (Fig. 5). Discussion Our results confirm the findings of others that it is possible to preserve lung allografts under hypothermic conditions for 24 hours.6, 13 Our study also indicates that a colloid hyperosmolar solution (MSGF) is a good medium for 24 hour lung preservation. There were no significant functional or structural differences between the fresh allografts and the lungs stored under hypothermic conditions in MSGF for up to 24 hours. Many methods of lung preservation have been used in the past. 1-13 Although it has proved effective for up to 72 hours in vitro,9 hypothermic pulsatile perfusion has not been tested in vivo. Other methods, such as hyperbaric oxygenation, have not been proved to be satisfactory in part because testing has not been adequate. Recently, one group6' 13 demonstrated that a hyperosmolar, intracellular crystalloid solution (Sacks solution) is a good medium for preservation of lung allografts under hypothermic conditions for up to 24 hours. When recipients of preserved lung allografts underwent immediate contralateral pulmonary artery ligation, no abnormal changes were observed in these allografts as
8 5 0 Toledo-Pereyra and Condie
The Journal of Thoracic and Cardiovascular Surgery
Fig. 4. Frontal (A) and lateral (B) plain chest roentgenograms of a dog 5 days after receiving a transplant of a lung preserved for 24 hours in MSGF (7° C ) . The right contralateral pulmonary artery has been ligated. There were no significant abnormalities seen in the lungs, except for a moderate infiltrate in the midfjeld of the right lung.
Fig. 5. Photomicrograph of a section of lung preserved fpr 24 hours in MSGF. This sample was taken 4 days after transplantation. Morphology is normal. (Hematoxylin and eosin. Original magnification (x375.)
Volume 76 Number 6 December, 1978
compared to control fresh allografts. It was not determined whether the important factors were related to hyperosmolarity, intracellular composition, or pH. Our experimental model in this study was the canine lung allograft, as in the studies by Veith and his associates. 13-16 Therefore, rejection and immunosuppression are limiting factors in testing the adequate functional response of the transplanted lung. The use of an autotransplant would obviously eliminate these factors. However, the difficulties involved in maintaining life in a pneumonectomized dog for long periods of time outweigh the potential advantages. Furthermore, technical factors, such as diminished length of pulmonary artery and left atrium suitable for anastomosis, make the use of the lung autograft model impractical for preservation studies. In any event, it is of considerable importance in lung preservation work to test fully the functional adequacy of the preserved lungs, as done by Kondo and associates5 with bilateral lung transplants or as done in the present work and a previous study by Veith's group13 with ligation of the opposite pulmonary artery. The exact mechanism for the beneficial effect of the colloid hyperosmolar solution is not known. It is possible that the hyperosmolarity, the high concentration of proteins, the high concentration of glucose, and the low concentrations of lipids or aggregable material play a significant role in extending long-term function of preserved allografts. On the other hand, it is worth noting that Veith and co-workers13 obtained similar results with a crystalloid, hyperosmolar intracellular solution. The common characteristics of both solutions, however, are attributable to different factors. In the MSGF solution, the albumin, glucose, and potassium contributed to the high osmolarity, while in the Sacks solution the hyperosmolarity was due to mannitol. Taking these findings into consideration, hyperosmolarity and hyperkalemia appear to be the most consistent factors in maintaining the viability of preserved lung allografts. Further studies in this area should clarify some of these theoretical implications. Although we have found that preservation at low temperatures (4 to 7° C.) is important, we do not think that hypothermia is, the most important fagtor in al{ cases, since 3 hours of hyppthermic preservation with a crystalloid extracellular type of solutjpn (Ringer's lactate) caused significant damage to preserved lung allografts. Hence, other factors besides hypothermia are also important. These factors are probably associated with the type of solution utilized during the period of storage, intracellular composition and hyperosmolarity
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being of utmost importance, as has been mentioned previously. In summary, these results confirm that lung preservation can be achieved for 24 hours with a colloid hyperosmolar solution (MSGF). A good response was observed after transplantation and immediate contralateral pulmonary artery ligation in recipients. REFERENCES 1 Blumenstock DA, Hechtman HB, Collins JA: Preservation of the canine lung. J THORAC CARDIOVASC SURG 44:771-775, 1962 2 Blumenstock DA, Lempert N, Morgado F: Preservation of the canine lung in vitro for 24 hours with the use of hypothermia and hyperbaric oxygen. J THORAC CARDIOVASC SURG 50:769-774, 1965 3 Castagna JT, Shors E, Benfield JR: The role of perfusion in lung preservation. J THORAC CARDIOVASC SURG 63:521-526, 1972 4 Connaughton PJ, Bahuth JJ, Lewis FJ: Lung ischemia up to six hours. Influence of local cooling in situ on subsequent pulmonary function. Chest 41:404-408, 1962 5 Kondo Y, Turner D, Cokrell JV, Hardy JD: Ischemic tolerance of the canine autotransplanted lung. Surgery 76:447-453, 1974 6 Merav AD, Crane R, Pinsker KL, Novin AJ, Koerner SK, Veith FJ: Preservation, transportation, and transplantation of lungs obtained after death. Surg Forum 28:195197, 1977 7 Stevens GH, Sanchez MN, Chappell G, Bennett LR, Gyepes MT, Fonkalsrud EW: Prolonged lung allograft preservation using inbred beagles. J Surg Res 12:246253, 1972 8 Toledo-Pereyra LH, Condie RM, Malmberg R, Simmons RL, Najarian JS: Long-term kidney preservation with a new plasma perfusate. Proc Clin Dial Transplant Forum 3:88-90, 1973 9 Toledo-Pereyra LH, Northrup WF, Humphrey ED, Najarian JS: Maintenance of lung viability for transplantation after long periods of hypothermic perfusion. J Surg Res 18:99-106, 1975 10 Toledo-Pereyra LH, Hau T, Simmons RL, Najarian JS: Lung preservation techniques. Ann Thorac Surg 23:487494, 1977 11 Toledo-Pergyra LH, Condie RM, Hau J, Simmons RL, Najarian JS; Three-day hyppthermic storage pf canine lungs. Surg Forum 28:19,4-19/7, 1977 12 Toledo-Pereyra LH, Simmons RL, Najarian JS: Factors determining successful liver preservation for transplantation. Ann Surg 181:289-298, 1975 13 Veith FJ, Crane R, Torres M, Colon I, Hagstrom JW, Pinsker K, Koerner SK: Effective preservation and transportation of lung transplants. J THORAC CARDIOVASC SURG 72:97-105, 1976
8 5 2 Toledo-Pereyra and Condie
14 VeithFJ, Richards K: Improved technique for canine lung transplantation. Ann Surg 171:553-558, 1970 15 Veith FJ, Sinha SBP, Siegelman SS, Hagstrom JWC: Single lung transplantation with immediate ligation of the opposite pulmonary artery in the dog. A model for assessing the functional adequacy of transplanted lungs, Research Animals in Medicine, Lt Harmison, ed., Bethesda,
The Journal of Thoracic and Cardiovascular Surgery
Md., 1973, Department of Health, Education, and Welfare Publication No. 72-333, pp 437-445 16 Veith FJ, Sinha SBP, Graves JS, Boley SJ, Daugherty JC: Ischemic tolerance of the lung. The effect of ventilation and inflation. J THORAC CARDIOVASC SURG 61:804810, 1971
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