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Successful 20 hour preservation of ischemic canine lung by hypothermia combined with minimal ventilation
Successful 20 hour preservation of ischémie canine lung by hypothermia combined with minimal ventilation Paul A. Thomas, M.D.,* and Robert J. Buchman, M.D., Colonel, MC,
USA,
Phoenixville, Pa.
I \ . practical limit for normothermic canine lung ischemia of about 2 hours has been established by experimentation, although considerable variance among individual ani mals has been observed.1· - The period of viable lung storage may be extended to 4 or more hours by hypothermie curtailment of cellular metabolism.3 Lung preservation during storage has been prolonged further by combining hypothermia with hyperbaria, ventilation, or vascular perfusion in various combinations. 40 This report summarizes a series of experiments directed toward exten sion of the period of lung storage, combin ing hypothermia with ventilation followed by successful transplantation of a viable organ to an immune-suppressed animal.
From the Department of Surgery, Thoracic Surgery Service, Valley Forge General Hospital, Phoenixville, Pa. 19460. Supported by Department of the Army Medical Research Grant No. 62110A 3A062110A826 00 106. The principles of laboratory animal care as established by the National Society for Medical Research were adhered to in these experiments. Received for publication Oct. 30, 1970. This material has been reviewed by the office of The Surgeon General, Department of the Army, and there is no objection to its presentation and/or publication. This review does not imply any indorsement of the opinions advanced or any recommendation of such products as may be named. ♦Present address, Division of Research, Lankenau Hospital, City Line and Lancaster Avenues, Philadelphia, Pa. 19151.
176
Materials and methods Experimental animals. Worm-free adult mongrel dogs of both sexes, weighing from 13 to 20 kilograms, were used for these experiments. The dogs were anesthetized by intravenous administration of sodium pentobarbital, 30 mg. per kilogram of body weight. Mechanically assisted ventilation with room air was provided through a cuffed endotracheal tube connected to a Harvard respirator. Aseptic precautions were used during the conduct of surgical procedures. Each animal subject was given intravenous fluids during surgical preparation, which included administration of 250 ml. of low molecular weight dextran (average molecular weight 40,000) immediately after restora tion of graft circulation. Chloramphenicol (500 mg.) was given to each animal daily. Surviving experimental animals were put to death 7 days after graft preparation by intra venous administration of a lethal dosage of pentobarbital. The left thorax was opened, and the lung was forcefully inflated via a preplaced endotracheal tube to inspect the gross appearance of the graft. The anasto moses were examined, and appropriate representative tissues were secured for preparation of histologie sections and sur face tension extracts. Lung preservation and storage. Immedi ately after excision of the proposed left
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T/ME
iHr
24 Hr
Fig. 1. Core temperature of the lung or lobe during preparation and placement in the preservation chamber. Temperature stability is maintained at 5° C. until removal just prior to restoration of circulation in the host animal.
lower lobe autograft or left lung allograft, the pulmonary artery was irrigated with a specially prepared, chilled (temperature 12° C.) perfusate. This was accomplished by gentle irrigation with a bulb syringe until the effluent solution was clear. The process re quired approximately 200 ml. of solution and reduced the lung temperature to 15° C. A 4 to 5 cm. length of tubular woven Dacron vascular prosthesis was circumferentially sutured to the bronchial stump for atraumatic airway cannulation to allow ven tilation of the lung during storage. Positivepressure ventilation with 100 to 150 ml. of room air was initiated, after which irriga tion through the pulmonary artery was re peated. The lobe or lung was then com pletely covered with coarse mesh gauze that was adjusted in a dependent position on the tray of the preservation chamber. The bot tom of the preservation tank was filled with enough perfusate to just cover the perforated tray so that the gauze would protect the graft specimen from drying out. The closed preservation chamber was placed in the bath of the Swenko* body organ preservation
unit. Ventilation of the stored lung was ad justed to 5 cycles per minute by connecting the Harvard respirator to the access tubing that exited through an opening in the chamber lid. The rate of lung cooling to a final storage temperature of 5° C. in the preservation chamber is depicted in Fig. 1. The following day, upon removal of the lung from storage, the bronchus was reexcised behind the suture anastomosis to the Dacron prosthesis. During the develop ment of storage techniques, it was observed that drying of the proximal bronchus and exposed carinae occurred frequently unless the prosthesis was of sufficient length and the gauze was fixed well above the anasto mosis to moisten incoming air during venti lation. The perfusate used for irrigation and storage of the excised lungs or lobes was prepared for that purpose by modifying the commercially distributed balanced electro lyte solution, lonosol T* with 5 per cent dextrose. To this basic solution, 1 million units of potassium penicillin G and sodium heparin 2.5 ml. (1,000 units per ml.) were
•Produced by Swenko Research and Development, Inc., Minneapolis, Minn.
♦Abbott Laboratories, North Chicago, 111.
The Journal of
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Thomas and Buchman
Thoracic and Cardiovascular Surgery
added. The solution was adjusted to an end pH of 7.3 by further adding 23 ml. of so dium bicarbonate (75 mg. per milliliter). Autograft procedure. Autografts were prepared by left lower lobectomy, storage, and reimplantation in 15 experimental ani mals. Six of these animals survived to be put to death after 7 days. From these ani mals, definitive data were derived. The tech nique described by Gago and colleagues7 was modified to meet the requirement for delayed placement of the autograft lobe until the day after excision. Immediately prior to occlusion of the lower lobe pulmo nary artery, regional anticoagulation was effected by local injection of heparinized 0.9 per cent sodium chloride solution. A generous cuff of left atrial tissue was excised with the inferior pulmonary vein, and the atrial defect was sutured. The lobe was pre pared and stored by the method described above. The following day, the left thorax of the experimental animal was re-entered and the pneumonectomy was completed. The tip of the left atrial appendage was am putated in preparation for everting suture anastomosis of the donor lobe atrial cuff to establish a venous drainage channel. Conti nuity of the pulmonary artery was re-estab lished, and a bronchial anastomosis was created. After ventilation of the autograft was initiated, the vascular occluding clamps were released to restore circulation. Allograft procedure. Left lung allografts were prepared in 13 experimental animals. Seven of these animals survived to be put to death 7 days later. Definitive data were derived from these animals. The surgical technique used was that described in prior communication from the laboratory.8 The donor animal was systemically anticoagulated by intravenous administration of heparin sodium (3 mg. per kilogram) 10 minutes before occlusion of the pulmonary artery. Generous lengths of the pulmonary artery and bronchus as well as a complete left atrial cuff were secured with the allo graft specimen. The donor dog was subse quently put to death. The left lung was pre pared and stored for 20 hours. The recipient
animal was readied by left pneumonectomy, with a maximum length of pulmonary artery and left bronchus preserved for anastomosis. The veins draining the left lung were indi vidually isolated, ligated, and divided to retain maximum atrial volume. The tip of the left atrial appendage was amputated as before to permit venous anastomosis with the donor atrial cuff in everting fashion. Pulmonary artery continuity was restored, and a bronchial anastomosis was created. Ventilation of the lung allograft was initiated prior to release of the occluding vascular clamps, which re-established circulation through the graft. Immune suppression. In these experi ments, suppression of the immune reaction to prevent allograft rejection in recipient animals was accomplished with a combina tion of azathioprine, prednisone, and antilymphocyte serum. Azathioprine was admin istered orally in a dosage of 7 mg. per kilo gram daily beginning the day of operation. Prednisone was also given orally, at a dos age of 20 mg. daily. Horse anti-canine lymphocyte serum* was administered by subcutaneous injection. Therapy with this agent was initiated the day prior to allo graft placement in an amount of 10 ml. per day for 2 days and 5 ml. per day thereafter for 6 days until sacrifice. In addition to the immune-suppressive drugs, all animals re ceived chloramphenicol to prevent compli cating pulmonary infection. Measurement of surface tension. The preparation of lung extracts and the method of measuring surface tension in this labora tory has been previously described in detail.8 The effects of hypothermia and minimal lung ventilation for the proposed period of stor age on pulmonary surfactant was not known prior to the initiation of these experiments. Therefore, the left lower lobes of lung were removed from 2 dogs, prepared for preser vation by the method outlined above, and placed in the storage chamber with ventila tion for 24 hours. These lungs were re* Obtained commercially from Microbiological Associates, Inc., Bethesda, Md.
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Table I. Surface tension of canine lung extracts after 24 hours preservation and storage at 5° C. with minimal ventilation A nimal No.
Surface Maximal
tension Minimal
(dynes/cm.) Index
20908
25
1
1.84
21303
28
9
1.02
moved from the chamber, and specimens were excised for extraction and measure ment of surface activity. Results The results of surface tension measure ments made of lung extracts that were pre pared after 24 hours of hypothermie pres ervation with minimal ventilation are presented in Table I. The surface activity of these extracts was normal. Therefore, the method of preparation and storage of the lungs or lobes used in these experiments was not expected to adversely influence pul monary surfactant. The results of the autograft experiments after 24 hours of storage, reimplantation, and 7 days survival of the recipient animals are presented in Table II. The surface ac tivity of extracts prepared from three of the recovered autografts was normal. The gross appearance of the in situ inflated lung specimens disclosed scattered areas of in farction surrounded by normal parenchyma (Fig. 2). The reason for this finding is not known with certainty; one may speculate that possibly the clearance of the pulmonary vasculature by irrigation prior to storage was not entirely satisfactory. Microscopic examination of histologie sections prepared from the grossly normal areas of lung re vealed nonpathologic parenchyma (Fig. 3). Healing of the donor side of the bronchial anastomosis was not uniform or satisfactory in all cases. The results of the allograft experiments after 20 hours of storage, transplantation, and 7 days survival of the recipient animals are presented in Table III. The surface ac tivity of extracts prepared from five of the
Fig. 2. Photograph of the inflated autograft lobe at the time of sacrifice of dog No. 21313. Note the predominant volume of normal appearing lung with one small area of infarction.
recovered allografts revealed insufficient surfactant activity in four and normal activ ity in one. Successful immune suppression was achieved in 2 animals, partially suc cessful suppression was realized in 4 ani mals, and no apparent beneficial effect was observed in 1 experimental animal. The gross appearance of the allografted lungs corresponded to the progress of the rejection reaction. The most rewarding result of this series was realized in experimental animal No. 23012. The allograft appeared grossly normal (Fig. 4 ) , as did the microscopic picture (Fig. 5). As anticipated, the pulmo nary surfactant content of this lung was also normal.
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Thoracic and Cardiovascular Surgery
Fig. 4. Photograph of the inflated allograft lung at the time of sacrifice of dog No. 23012. Grossly, this lung appears normal.
Fig. 5. Photomicrograph of the lung from dog No. 23012. The lung is normal, without any evidence of rejection 1 week after preparation. (Original magnification xlOO.)
Table II. Results of reimplantation experiments Surface tension of lung extract (dynes/cm.) Animal
No.
17959 18746 18996 21214 21313 21946
Minimal
Pathology
S*
3 5 1
1.56 1.40 1.84
Gross
Microscopic
Patchy infarctions Lung normal, bronchus Normal Lung normal, bronchus Normal Lung normal, bronchus Patchy infarctions Lung normal, bronchus Small infarct Lung normal, bronchus Small infarct Lung normal, bronchus
not healed healed not healed healed healed healed
*S, Stability index calculated as 2(maximal S.T. - minimal S.T.) / maximal S.T. + minimal S.T.
Table III. Results of transphntation
experiments
Surface ension of lung extract (dynes/cm.) Animal
No.
203012 25117 25285 23641 25928 26277 8620
Pathology
S*
Gross
Microscopic
8 14 16
1.14 0.83 0.70
16 10
0.51 0.92
Normal Patchy infarcts Patchy infarcts Infarcted Patchy edema Patchy infarcts Infarcted
No rejection, normal lung N o rejection, partial atelectasis Minimal rejection, atelectasis Severe rejection Moderate rejection Moderate rejection Severe rejection
Minimal
*S, Stability index calculated as 2(maximal S.T. - minimal S.T.) / maximal S.T. + minimal S.T.
Discussion At the present time, a sense of urgency to re-establish circulation from the proposed recipient through the donor organ to avoid ischémie injury attends the acquisition effort. It is usually considered expedient to pre pare the recipient candidate by excision of
the destroyed or defective organ simulta neously with the initiation of the removal of a donor organ. This may easily become a problem of overwhelming proportions if multiple organs are to be harvested and used in several recipient patients. Donor organ protection during temporary periods
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of ischemia is afforded by the application of hypothermia, which may be initiated prior to removal and maintained several hours until circulation is re-established. A longer period of reliable, non-injurious preserva tion is desirable to support a practical organ transplantation program. This time is needed to permit optimum selection of genetically suitable recipients and to prepare them for the surgical ordeal. Brownlee and Couves3 reported that lung preservation at 4 to 10° C. was reliable up to 4 hours, as demonstrated by in vivo per fusion and gas exchange occurring in the autograft. Garzon and colleagues4 achieved successful 23 to 26 hour lung preservation by the addition of a hyperbaric (2 atm.) oxygen atmosphere to hypothermia. They tested the in vivo gas exchange function of the lung autograph serially for extended periods. Their persistent efforts were re warded by the survival of a significant group of experimental animals; however, the pres ervation method used fell short of the required reliability necessary for clinical ap plication. Homatas and associates2 observed a beneficial effect of continued lung ventila tion up to the time of harvest. This observa tion confirms the much earlier experimental results reported by Blumenstock and coworkers," who noted that minimal ventila tion during storage was beneficial. In addi tion to keeping the alveoli in a most natural attitude, the introduction of diffusible ox ygen may provide for the minimal metabolic requirements of the hypothermie lung. An other remarkable feature of the study con ducted by Blumenstock and colleagues" was their ability to achieve success by trans planting the stored lung into an immunesuppressed host animal. Several alternative methods of preparing and storing lungs were explored without notable success initially. During this period of development, the advantage of using the left lower lobe as an autograft became ap parent. Removal of this anatomic unit of the canine lung is readily accomplished, pre serving adequate length of the pulmonary artery and bronchus as well as a cuff of
the atrium with the inferior pulmonary vein. The experimental animal thereby is given an improved chance of surviving the initial operation followed by a second opening of the chest 24 hours later without accident. After completing the left pneumonectomy, it is technically easy to reimplant the stored lower lobe. This technique eliminated re jection as a complication in the evaluation of results. The selection of 24 hours as the storage period for the autograft experiments was purely arbitrary. Although the reimplanted lobes were nearly normal and alveolar cell metabolism was sufficiently ac tive to produce pulmonary surfactant, heal ing of the bronchus was not uniformly satis factory on the autograft side. The results achieved in the series of lung autograft experiments were encouraging; therefore, the allografts were undertaken. The time of storage was electively shortened to 20 hours to improve the probability of preserving viability. As anticipated, immune rejection was a prominent feature in this series of experimental animals. The effective ness of the immune-suppressive therapy was comparable to that achieved in other ani mals which were subjected to left lung allografts without storage in this laboratory. Obviously, organ viability during storage was preserved; otherwise, rejection would not have progressed in these lungs to the degree observed. However, pulmonary edema appeared with distressing frequency within 1 or 2 hours after circulation through the graft was re-established. Edema sub sided spontaneously in several hours, but it may be indicative of some reversible cellular injury resulting from the prolonged period of storage. This complication was also ob served and remarked upon by Garzon and colleagues.4 It is apparent that the cost of additional storage time will be the require ment for continuous lung perfusion or the application of organ freezing techniques. Venous obstruction by suture line throm bosis of the atrial anastomosis has been the foremost complication of experimental lung transplantation. Some investigators4 who have reported improved results have ob-
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Thomas and Buchman
Thoracic and Cardiovascular Surgery
viated this complication by anticoagulation of recipient animals. We have attempted to minimize this complication in the immediate postautograft or -allograft period by admin istration of low molecular weight dextran to the recipient animals. Summary Preservation of viability of canine lung lobes during storage for 24 hours has been achieved with hypothermia, 5° C , and mini mal ventilation. The presence of normal amounts of pulmonary surfactant in the autografted lung lobes, as well as the normal histologie appearance observed, indicates that the stored lobes were contributing some respiratory function for the experimental animal up to the time of sacrifice 1 week after preparation. Extension of these ex periments to include storage and transplan tation of the lung to a new host was ac complished, but with notably less success. The additional challenge of providing suit able immune suppression for the recipient animal to avert rejection of the stored allo graft was comparable to that encountered in previous transplantation experiments with freshly acquired lung. However, the demon strated, albiet limited, success of this effort after 20 hours of allograft storage is signifi cant in the further development of tech niques that will be applicable to a practical lung transplantation program.
REFERENCES 1 Veith, F . J., Koerner, S., Lari Attai, M. T., and Gliedman, M . L.: Functional Evaluation of Lungs Transplanted After Varying Ischémie Intervals and After Preservation by Mechanical Ventricular Assistance, Ann. Thorac. Surg. 7: 446-458, 1969. 2 Homatas, J., Bryant, L., and Eiseman, B.: Time Limits of Cadaver Lung Viability, J.
THORAC.
CARDIOVASC.
SURG.
56:
132-140,
1968. 3 Brownlee, R. T., and Couves, C. M . : Factors Concerned in the Maintenance of Viability in Pulmonary Transplants, Ann. Thorac. Surg. 5: 112-121, 1968. 4 Garzon, A. A., Cheng, C , Pangan, J., and Karlsen, K. E.: Hypothermie Hyperbaric Lung Preservation for Twenty-Four Hours With Re plantation,
J. T H O R A C
CARDIOVASC.
SURG. 55:
546-554, 1968. 5 Largiader, F., Manax, W. G., Lyons, G. W., and Lillehei, R. C : Technical Aspects of Trans plantation of Preserved Lungs, Dis. Chest 49: 1-7, 1966. 6 Blumenstock, D . A., Hechtman, H. B., and Collins, J. A.: Preservation of the Canine Lung, J.
THORAC.
CARDIOVASC.
SURG.
44:
770-775,
1962. 7 Gago, O., Delgado, E., Archer, F . L., Schoenfeld, F . G., Ranniger, K., Nigro, S. L., and Adams, W. E . : Homotransplantation and Autotransplantation of a Pulmonary Lobe, J. THORAC CARDIOVASC SURG. 48: 726-732 1964.
8 Thomas, P. A., and Jolly, P. C : Preservation of Pulmonary Surfactant Activity in Canine Lung Allografts by Immune Suppressive Therapy,
J.
THORAC
405-410, 1968.
CARDIOVASC
SURG.
55:
USA,
Phoenixville, Pa.
I \ . practical limit for normothermic canine lung ischemia of about 2 hours has been established by experimentation, although considerable variance among individual ani mals has been observed.1· - The period of viable lung storage may be extended to 4 or more hours by hypothermie curtailment of cellular metabolism.3 Lung preservation during storage has been prolonged further by combining hypothermia with hyperbaria, ventilation, or vascular perfusion in various combinations. 40 This report summarizes a series of experiments directed toward exten sion of the period of lung storage, combin ing hypothermia with ventilation followed by successful transplantation of a viable organ to an immune-suppressed animal.
From the Department of Surgery, Thoracic Surgery Service, Valley Forge General Hospital, Phoenixville, Pa. 19460. Supported by Department of the Army Medical Research Grant No. 62110A 3A062110A826 00 106. The principles of laboratory animal care as established by the National Society for Medical Research were adhered to in these experiments. Received for publication Oct. 30, 1970. This material has been reviewed by the office of The Surgeon General, Department of the Army, and there is no objection to its presentation and/or publication. This review does not imply any indorsement of the opinions advanced or any recommendation of such products as may be named. ♦Present address, Division of Research, Lankenau Hospital, City Line and Lancaster Avenues, Philadelphia, Pa. 19151.
176
Materials and methods Experimental animals. Worm-free adult mongrel dogs of both sexes, weighing from 13 to 20 kilograms, were used for these experiments. The dogs were anesthetized by intravenous administration of sodium pentobarbital, 30 mg. per kilogram of body weight. Mechanically assisted ventilation with room air was provided through a cuffed endotracheal tube connected to a Harvard respirator. Aseptic precautions were used during the conduct of surgical procedures. Each animal subject was given intravenous fluids during surgical preparation, which included administration of 250 ml. of low molecular weight dextran (average molecular weight 40,000) immediately after restora tion of graft circulation. Chloramphenicol (500 mg.) was given to each animal daily. Surviving experimental animals were put to death 7 days after graft preparation by intra venous administration of a lethal dosage of pentobarbital. The left thorax was opened, and the lung was forcefully inflated via a preplaced endotracheal tube to inspect the gross appearance of the graft. The anasto moses were examined, and appropriate representative tissues were secured for preparation of histologie sections and sur face tension extracts. Lung preservation and storage. Immedi ately after excision of the proposed left
Volume 62
Preservation of ischémie lung
Number 2
17 7
August, 1971
T/ME
iHr
24 Hr
Fig. 1. Core temperature of the lung or lobe during preparation and placement in the preservation chamber. Temperature stability is maintained at 5° C. until removal just prior to restoration of circulation in the host animal.
lower lobe autograft or left lung allograft, the pulmonary artery was irrigated with a specially prepared, chilled (temperature 12° C.) perfusate. This was accomplished by gentle irrigation with a bulb syringe until the effluent solution was clear. The process re quired approximately 200 ml. of solution and reduced the lung temperature to 15° C. A 4 to 5 cm. length of tubular woven Dacron vascular prosthesis was circumferentially sutured to the bronchial stump for atraumatic airway cannulation to allow ven tilation of the lung during storage. Positivepressure ventilation with 100 to 150 ml. of room air was initiated, after which irriga tion through the pulmonary artery was re peated. The lobe or lung was then com pletely covered with coarse mesh gauze that was adjusted in a dependent position on the tray of the preservation chamber. The bot tom of the preservation tank was filled with enough perfusate to just cover the perforated tray so that the gauze would protect the graft specimen from drying out. The closed preservation chamber was placed in the bath of the Swenko* body organ preservation
unit. Ventilation of the stored lung was ad justed to 5 cycles per minute by connecting the Harvard respirator to the access tubing that exited through an opening in the chamber lid. The rate of lung cooling to a final storage temperature of 5° C. in the preservation chamber is depicted in Fig. 1. The following day, upon removal of the lung from storage, the bronchus was reexcised behind the suture anastomosis to the Dacron prosthesis. During the develop ment of storage techniques, it was observed that drying of the proximal bronchus and exposed carinae occurred frequently unless the prosthesis was of sufficient length and the gauze was fixed well above the anasto mosis to moisten incoming air during venti lation. The perfusate used for irrigation and storage of the excised lungs or lobes was prepared for that purpose by modifying the commercially distributed balanced electro lyte solution, lonosol T* with 5 per cent dextrose. To this basic solution, 1 million units of potassium penicillin G and sodium heparin 2.5 ml. (1,000 units per ml.) were
•Produced by Swenko Research and Development, Inc., Minneapolis, Minn.
♦Abbott Laboratories, North Chicago, 111.
The Journal of
17 8
Thomas and Buchman
Thoracic and Cardiovascular Surgery
added. The solution was adjusted to an end pH of 7.3 by further adding 23 ml. of so dium bicarbonate (75 mg. per milliliter). Autograft procedure. Autografts were prepared by left lower lobectomy, storage, and reimplantation in 15 experimental ani mals. Six of these animals survived to be put to death after 7 days. From these ani mals, definitive data were derived. The tech nique described by Gago and colleagues7 was modified to meet the requirement for delayed placement of the autograft lobe until the day after excision. Immediately prior to occlusion of the lower lobe pulmo nary artery, regional anticoagulation was effected by local injection of heparinized 0.9 per cent sodium chloride solution. A generous cuff of left atrial tissue was excised with the inferior pulmonary vein, and the atrial defect was sutured. The lobe was pre pared and stored by the method described above. The following day, the left thorax of the experimental animal was re-entered and the pneumonectomy was completed. The tip of the left atrial appendage was am putated in preparation for everting suture anastomosis of the donor lobe atrial cuff to establish a venous drainage channel. Conti nuity of the pulmonary artery was re-estab lished, and a bronchial anastomosis was created. After ventilation of the autograft was initiated, the vascular occluding clamps were released to restore circulation. Allograft procedure. Left lung allografts were prepared in 13 experimental animals. Seven of these animals survived to be put to death 7 days later. Definitive data were derived from these animals. The surgical technique used was that described in prior communication from the laboratory.8 The donor animal was systemically anticoagulated by intravenous administration of heparin sodium (3 mg. per kilogram) 10 minutes before occlusion of the pulmonary artery. Generous lengths of the pulmonary artery and bronchus as well as a complete left atrial cuff were secured with the allo graft specimen. The donor dog was subse quently put to death. The left lung was pre pared and stored for 20 hours. The recipient
animal was readied by left pneumonectomy, with a maximum length of pulmonary artery and left bronchus preserved for anastomosis. The veins draining the left lung were indi vidually isolated, ligated, and divided to retain maximum atrial volume. The tip of the left atrial appendage was amputated as before to permit venous anastomosis with the donor atrial cuff in everting fashion. Pulmonary artery continuity was restored, and a bronchial anastomosis was created. Ventilation of the lung allograft was initiated prior to release of the occluding vascular clamps, which re-established circulation through the graft. Immune suppression. In these experi ments, suppression of the immune reaction to prevent allograft rejection in recipient animals was accomplished with a combina tion of azathioprine, prednisone, and antilymphocyte serum. Azathioprine was admin istered orally in a dosage of 7 mg. per kilo gram daily beginning the day of operation. Prednisone was also given orally, at a dos age of 20 mg. daily. Horse anti-canine lymphocyte serum* was administered by subcutaneous injection. Therapy with this agent was initiated the day prior to allo graft placement in an amount of 10 ml. per day for 2 days and 5 ml. per day thereafter for 6 days until sacrifice. In addition to the immune-suppressive drugs, all animals re ceived chloramphenicol to prevent compli cating pulmonary infection. Measurement of surface tension. The preparation of lung extracts and the method of measuring surface tension in this labora tory has been previously described in detail.8 The effects of hypothermia and minimal lung ventilation for the proposed period of stor age on pulmonary surfactant was not known prior to the initiation of these experiments. Therefore, the left lower lobes of lung were removed from 2 dogs, prepared for preser vation by the method outlined above, and placed in the storage chamber with ventila tion for 24 hours. These lungs were re* Obtained commercially from Microbiological Associates, Inc., Bethesda, Md.
Volume 62
Preservation of ischémie lung
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August, 1971
Table I. Surface tension of canine lung extracts after 24 hours preservation and storage at 5° C. with minimal ventilation A nimal No.
Surface Maximal
tension Minimal
(dynes/cm.) Index
20908
25
1
1.84
21303
28
9
1.02
moved from the chamber, and specimens were excised for extraction and measure ment of surface activity. Results The results of surface tension measure ments made of lung extracts that were pre pared after 24 hours of hypothermie pres ervation with minimal ventilation are presented in Table I. The surface activity of these extracts was normal. Therefore, the method of preparation and storage of the lungs or lobes used in these experiments was not expected to adversely influence pul monary surfactant. The results of the autograft experiments after 24 hours of storage, reimplantation, and 7 days survival of the recipient animals are presented in Table II. The surface ac tivity of extracts prepared from three of the recovered autografts was normal. The gross appearance of the in situ inflated lung specimens disclosed scattered areas of in farction surrounded by normal parenchyma (Fig. 2). The reason for this finding is not known with certainty; one may speculate that possibly the clearance of the pulmonary vasculature by irrigation prior to storage was not entirely satisfactory. Microscopic examination of histologie sections prepared from the grossly normal areas of lung re vealed nonpathologic parenchyma (Fig. 3). Healing of the donor side of the bronchial anastomosis was not uniform or satisfactory in all cases. The results of the allograft experiments after 20 hours of storage, transplantation, and 7 days survival of the recipient animals are presented in Table III. The surface ac tivity of extracts prepared from five of the
Fig. 2. Photograph of the inflated autograft lobe at the time of sacrifice of dog No. 21313. Note the predominant volume of normal appearing lung with one small area of infarction.
recovered allografts revealed insufficient surfactant activity in four and normal activ ity in one. Successful immune suppression was achieved in 2 animals, partially suc cessful suppression was realized in 4 ani mals, and no apparent beneficial effect was observed in 1 experimental animal. The gross appearance of the allografted lungs corresponded to the progress of the rejection reaction. The most rewarding result of this series was realized in experimental animal No. 23012. The allograft appeared grossly normal (Fig. 4 ) , as did the microscopic picture (Fig. 5). As anticipated, the pulmo nary surfactant content of this lung was also normal.
The Journal of
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Thomas and Buchman
Thoracic and Cardiovascular Surgery
Fig. 4. Photograph of the inflated allograft lung at the time of sacrifice of dog No. 23012. Grossly, this lung appears normal.
Fig. 5. Photomicrograph of the lung from dog No. 23012. The lung is normal, without any evidence of rejection 1 week after preparation. (Original magnification xlOO.)
Table II. Results of reimplantation experiments Surface tension of lung extract (dynes/cm.) Animal
No.
17959 18746 18996 21214 21313 21946
Minimal
Pathology
S*
3 5 1
1.56 1.40 1.84
Gross
Microscopic
Patchy infarctions Lung normal, bronchus Normal Lung normal, bronchus Normal Lung normal, bronchus Patchy infarctions Lung normal, bronchus Small infarct Lung normal, bronchus Small infarct Lung normal, bronchus
not healed healed not healed healed healed healed
*S, Stability index calculated as 2(maximal S.T. - minimal S.T.) / maximal S.T. + minimal S.T.
Table III. Results of transphntation
experiments
Surface ension of lung extract (dynes/cm.) Animal
No.
203012 25117 25285 23641 25928 26277 8620
Pathology
S*
Gross
Microscopic
8 14 16
1.14 0.83 0.70
16 10
0.51 0.92
Normal Patchy infarcts Patchy infarcts Infarcted Patchy edema Patchy infarcts Infarcted
No rejection, normal lung N o rejection, partial atelectasis Minimal rejection, atelectasis Severe rejection Moderate rejection Moderate rejection Severe rejection
Minimal
*S, Stability index calculated as 2(maximal S.T. - minimal S.T.) / maximal S.T. + minimal S.T.
Discussion At the present time, a sense of urgency to re-establish circulation from the proposed recipient through the donor organ to avoid ischémie injury attends the acquisition effort. It is usually considered expedient to pre pare the recipient candidate by excision of
the destroyed or defective organ simulta neously with the initiation of the removal of a donor organ. This may easily become a problem of overwhelming proportions if multiple organs are to be harvested and used in several recipient patients. Donor organ protection during temporary periods
Volume 62 Number 2
Preservation of ischémie lung
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of ischemia is afforded by the application of hypothermia, which may be initiated prior to removal and maintained several hours until circulation is re-established. A longer period of reliable, non-injurious preserva tion is desirable to support a practical organ transplantation program. This time is needed to permit optimum selection of genetically suitable recipients and to prepare them for the surgical ordeal. Brownlee and Couves3 reported that lung preservation at 4 to 10° C. was reliable up to 4 hours, as demonstrated by in vivo per fusion and gas exchange occurring in the autograft. Garzon and colleagues4 achieved successful 23 to 26 hour lung preservation by the addition of a hyperbaric (2 atm.) oxygen atmosphere to hypothermia. They tested the in vivo gas exchange function of the lung autograph serially for extended periods. Their persistent efforts were re warded by the survival of a significant group of experimental animals; however, the pres ervation method used fell short of the required reliability necessary for clinical ap plication. Homatas and associates2 observed a beneficial effect of continued lung ventila tion up to the time of harvest. This observa tion confirms the much earlier experimental results reported by Blumenstock and coworkers," who noted that minimal ventila tion during storage was beneficial. In addi tion to keeping the alveoli in a most natural attitude, the introduction of diffusible ox ygen may provide for the minimal metabolic requirements of the hypothermie lung. An other remarkable feature of the study con ducted by Blumenstock and colleagues" was their ability to achieve success by trans planting the stored lung into an immunesuppressed host animal. Several alternative methods of preparing and storing lungs were explored without notable success initially. During this period of development, the advantage of using the left lower lobe as an autograft became ap parent. Removal of this anatomic unit of the canine lung is readily accomplished, pre serving adequate length of the pulmonary artery and bronchus as well as a cuff of
the atrium with the inferior pulmonary vein. The experimental animal thereby is given an improved chance of surviving the initial operation followed by a second opening of the chest 24 hours later without accident. After completing the left pneumonectomy, it is technically easy to reimplant the stored lower lobe. This technique eliminated re jection as a complication in the evaluation of results. The selection of 24 hours as the storage period for the autograft experiments was purely arbitrary. Although the reimplanted lobes were nearly normal and alveolar cell metabolism was sufficiently ac tive to produce pulmonary surfactant, heal ing of the bronchus was not uniformly satis factory on the autograft side. The results achieved in the series of lung autograft experiments were encouraging; therefore, the allografts were undertaken. The time of storage was electively shortened to 20 hours to improve the probability of preserving viability. As anticipated, immune rejection was a prominent feature in this series of experimental animals. The effective ness of the immune-suppressive therapy was comparable to that achieved in other ani mals which were subjected to left lung allografts without storage in this laboratory. Obviously, organ viability during storage was preserved; otherwise, rejection would not have progressed in these lungs to the degree observed. However, pulmonary edema appeared with distressing frequency within 1 or 2 hours after circulation through the graft was re-established. Edema sub sided spontaneously in several hours, but it may be indicative of some reversible cellular injury resulting from the prolonged period of storage. This complication was also ob served and remarked upon by Garzon and colleagues.4 It is apparent that the cost of additional storage time will be the require ment for continuous lung perfusion or the application of organ freezing techniques. Venous obstruction by suture line throm bosis of the atrial anastomosis has been the foremost complication of experimental lung transplantation. Some investigators4 who have reported improved results have ob-
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Thoracic and Cardiovascular Surgery
viated this complication by anticoagulation of recipient animals. We have attempted to minimize this complication in the immediate postautograft or -allograft period by admin istration of low molecular weight dextran to the recipient animals. Summary Preservation of viability of canine lung lobes during storage for 24 hours has been achieved with hypothermia, 5° C , and mini mal ventilation. The presence of normal amounts of pulmonary surfactant in the autografted lung lobes, as well as the normal histologie appearance observed, indicates that the stored lobes were contributing some respiratory function for the experimental animal up to the time of sacrifice 1 week after preparation. Extension of these ex periments to include storage and transplan tation of the lung to a new host was ac complished, but with notably less success. The additional challenge of providing suit able immune suppression for the recipient animal to avert rejection of the stored allo graft was comparable to that encountered in previous transplantation experiments with freshly acquired lung. However, the demon strated, albiet limited, success of this effort after 20 hours of allograft storage is signifi cant in the further development of tech niques that will be applicable to a practical lung transplantation program.
REFERENCES 1 Veith, F . J., Koerner, S., Lari Attai, M. T., and Gliedman, M . L.: Functional Evaluation of Lungs Transplanted After Varying Ischémie Intervals and After Preservation by Mechanical Ventricular Assistance, Ann. Thorac. Surg. 7: 446-458, 1969. 2 Homatas, J., Bryant, L., and Eiseman, B.: Time Limits of Cadaver Lung Viability, J.
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56:
132-140,
1968. 3 Brownlee, R. T., and Couves, C. M . : Factors Concerned in the Maintenance of Viability in Pulmonary Transplants, Ann. Thorac. Surg. 5: 112-121, 1968. 4 Garzon, A. A., Cheng, C , Pangan, J., and Karlsen, K. E.: Hypothermie Hyperbaric Lung Preservation for Twenty-Four Hours With Re plantation,
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546-554, 1968. 5 Largiader, F., Manax, W. G., Lyons, G. W., and Lillehei, R. C : Technical Aspects of Trans plantation of Preserved Lungs, Dis. Chest 49: 1-7, 1966. 6 Blumenstock, D . A., Hechtman, H. B., and Collins, J. A.: Preservation of the Canine Lung, J.
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1962. 7 Gago, O., Delgado, E., Archer, F . L., Schoenfeld, F . G., Ranniger, K., Nigro, S. L., and Adams, W. E . : Homotransplantation and Autotransplantation of a Pulmonary Lobe, J. THORAC CARDIOVASC SURG. 48: 726-732 1964.
8 Thomas, P. A., and Jolly, P. C : Preservation of Pulmonary Surfactant Activity in Canine Lung Allografts by Immune Suppressive Therapy,
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