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Ann Thorac Surg 1996;61:1488-1493
© 1996 The Society of Thoracic Surgeons
Department of Pathology and Laboratory Medicine, Pathology Service VA Medical Center, and Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky; Department of Surgery, University of Michigan, Ann Arbor, Michigan; and National Institute on Drug Abuse Addiction Research Center, Baltimore, Maryland
Accepted for publication January 22, 1996.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. In this study, we examined the effect of infusing plasma containing the hibernation induction trigger molecule on lung preservation for transplantation using a multiorgan autoperfusion block.
Results. This study demonstrated that successful orthotopic transplantation of single canine lungs is possible after 24 to 33 hours of preservation when the lung has been maintained with plasma containing the hibernation induction trigger molecule.
Conclusions. Theoretically, hibernation induction trigger could be administered to donors before lung harvest in an effort to extend lung preservation times.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We have previously demonstrated that an albumin fraction derived from hibernating animals contains a potent metabolic inhibitor known as the hibernation induction trigger (HIT) [7]. By infusing primates (Maccaca mulata) with this HIT-active fraction, we induced profound behavioral and physiologic depression resembling a natural hibernation state including the appearance of an anesthetized state, hypothermia, bradycardia, decreased renal function, and long-term feeding inhibition. Moreover, all the aforementioned behavioral and physiologic changes could be reversed or retarded by the infusion of opiate antagonists naloxone and naltrexone [8–10]. Such evidence indicated that the HIT molecule may initiate its potent metabolic inhibitory effects in nonhibernating recipients through specific peripheral and central opioid receptors.
Furthermore, we recently reported the results of organ preservation using a multiorgan autoperfusion model consisting of the heart/lungs, kidneys, liver, pancreas, and a short portion of the duodenum. In this model, organs could be preserved for 18 to 37 hours (average, 24.6 ± 2.7 hours) [11–13]. Interestingly, however, within a short time, lungs from these blocks were unsuitable for transplantation because of ventilatory failure and microthrombi formation.
In succeeding experiments, organ preservation time was markedly extended when 10 mL of plasma containing the HIT factor obtained from deeply hibernating woodchucks was infused intravenously 2 hours before the operation and 4 mL at 4-hour intervals during the preservation period. Organ block survival time ranged from 33 to 56 hours (mean, 43.4 ± 4.1 hours), and lung damage appeared to be markedly reduced [14].
In a succeeding study, the delta opioid DADLE (D-Ala2-Leu-5-enkephalin), which mimics the activity of the HIT molecule in inducing summer hibernation in ground squirrels, was infused at 1 mg/kg before organ block harvesting and at 2-hour intervals during the preservation period. Survival time averaged 41 to 60 hours with a mean of 46.6 hours [15]. In this article, we report the results of canine single-lung transplantation after more than 24 hours of preservation using the multiorgan autoperfusion block model treated with infusion of HIT.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Pretreatment
All donor dogs were given 2 g of neomycin orally once a day for 3 days before operation to reduce gut flora. All dogs were fasted for 10 hours preoperatively. After the administration of anesthetics, donor dogs received 10 mL of HIT-containing plasma intravenously.
Sample Procurement
Plasma assayed for HIT activity was obtained from woodchucks weighing 3.0 to 5.0 kg, which were maintained in a hibernaculum at 4° to 6°C during the winter months. Blood was drawn aseptically by intraventricular puncture while each animal was in deep hibernation, as evidenced by a core temperature of approximately 5°C and a heart rate ranging from 1 to 2 beats/min. The whole blood was placed in heparinized tubes and centrifuged to obtain plasma, which was then frozen at -70°C for later use.
The surgical technique used to harvest the organs was similar to that previously reported [11]. A brief review of this technique follows.
Multiorgan Block Preservation Model
The bath solution was prewarmed and maintained at 32°C ± 1°C. Mechanical ventilation was maintained by a Harvard Ventilator. Tidal volume of 500 to 700 mL, rate of 10 to 20 rpm, and positive end-expiratory pressure of 4 to 8 mm Hg were maintained. A gas mixture of 50% O2 + 3% CO2 + 47% N2 was delivered via the ventilator. A 5% dextrose solution containing the following drugs was infused through the portal vein at 10 to 20 mL/h: calcium chloride (1 g/L), insulin (50 units/L), mannitol (12.5 g/L), methylprednisolone (500 mg/L), penicillin (1,000,000 units/L), and Flagyl (metronidazole hydrochloride; Sciapparelli Searle, Chicago, IL; 500 mg/L). A fat emulsion (Soyacal; Alpha Therapeutics Corp, Los Angeles, CA; 2 mL) and methylprednisolone (30 mg) were injected through the portal vein every 2 hours. Blood transfusions were given as needed to maintain aortic systolic pressure between 75 and 100 mm Hg and central venous pressure between 0 and 10 mm Hg. Plasma was given instead of whole blood if the hematocrit was greater than 45%. Four milliliters of the HIT-containing plasma from hibernating woodchucks was infused through the portal vein every 4 hours during the preservation period.
Monitoring
Aortic pressure, central venous pressure, and portal venous pressure were monitored and recorded on a Gould Multichannel Recorder (Gould Inc, Centerville, OH) throughout the preservation period. Blood gas measurements were performed every hour using an IL Blood Gas-Electrolyte Analyzer (Instrumentation Laboratory, Lexington, MA), or continuously monitored using PB-3300 Blood Gas Monitor (Puriton-Bennett, Anaheim, CA). Respiratory pressure, tidal volume, and positive end-expiratory pressure for the lungs were recorded every hour, as were observable changes in the lungs, such as color, atelectasis, air leaking, and edema. Blood samples were also obtained every 4 hours for hematologic analysis and biochemical analysis including liver, kidney, and pancreatic function tests as well as heart isoenzyme determinations. Temperature, urine and bile production, and duodenal and pancreatic secretions were monitored and recorded every hour.
Determination of Tissue Wet/Dry Weight Ratio
At the termination of the experiment, tissue specimens were taken from each organ for tissue wet/dry ratio determination. Tissue samples were blotted to remove excess fluid, and wet weight was measured. The dry weight was determined after the samples had been dried in an oven at 85°C for 72 hours.
Statistical Analysis
All laboratory test results obtained before the experimental procedure (blood gases, hematocrit, blood chemistries, hematology, lactic acid, and enzymes for heart, liver, pancreas, and kidney functions) were used as normal controls. These controls were compared with the results obtained during the preservation period. Hemodynamic values and urine output were measured immediately after harvesting and during the preservation period, and these results were compared. Tissue wet/dry weight ratios for all the organs were compared with those obtained from normal dogs.
For comparisons within a group, analysis of variance and Student-Newman-Keuls tests were used to compare the data measured during the preservation period with those obtained preoperatively. If a comparison was needed between the study group and the control group at a certain point, an unpaired Student's t test was used. All data are expressed as mean ± standard error of the mean, with statistical significance assigned at p less than 0.05.
Lung Transplantation From Preserved Organ Block
ORGAN BLOCK-DONOR PROCEDURE.
In the preservation block, 5,000 µg of heparin sodium was infused into the venous line. Dissection of the left lung was performed to expose the left pulmonary artery, the left main bronchus, and the left atrium, as in the recipient. A transfusion cannula was inserted into the left pulmonary artery through the main pulmonary artery, and cold Collins solution was administered. The left atrium was opened for fluid drainage. After 1,000 mL of preservation solution had been infused and the lung tissue was cold, the left lung was removed with the pulmonary artery, the left main bronchus, and the left atrium. The removed left lung was wrapped in an ice-cooled wet towel and placed in the recipient for anastomosis.
RECIPIENT PROCEDURE.
The times of preservation were 24 hours (3 dogs), 25 hours (1 dog), 30 hours (1 dog), and 33 hours (1 dog), averaging 26.7 ± 1.4 hours. The transplantation technique was a modification of that reported by Veith and Richards [16]. The dog was anesthetized with 30 mg/kg of sodium pentobarbital, intubated, and artificially ventilated. The chest was opened and the left pulmonary artery was dissected free. The pericardium was opened over the main pulmonary artery. The right main pulmonary artery was dissected free, and a 10-mm IVM OC hydraulic vascular occluder (In Vivo Metric, Healdsburg, CA) was sutured around it for later occlusion. The posterior main bronchus was separated from the left atrium. The left pulmonary artery was divided near its first branch. The left main bronchus was occluded with an angled atraumatic clamp and divided proximal to the origin of the upper lobe.
The left atrium was further mobilized by dividing the fat and visceral pericardium along the superior border of the left atrium. An angled atraumatic clamp was placed across the left atrium as far medially as possible without occluding the right inferior pulmonary vein. The left pulmonary veins were then transected at their junction with the left atrium, and the intervening tissue was incised over a clamp. The left lung was removed.
Both ends of the left atrium were anastomosed using two everting mattress sutures of 4-0 Prolene (Ethicon, Somerville, NJ). Air was flushed out with saline solution before the sutures were tied off. Next, the bronchial anastomosis was performed. Two 3-0 Prolene sutures were used for end-to-end anastomosis. After the bronchial anastomosis was completed, the bronchial clamp was removed, allowing expansion and ventilation of the transplanted left lung.
The donor and recipient pulmonary arteries were anastomosed with continuous 4-0 Prolene sutures. Saline solution was used to fill the artery and expel air before the last stitch. The clamps were released, and any leaks were repaired. The chest was closed in layers, and the hydraulic occluder was brought out through the incision.
Treatment of the Recipient Animals After Transplantation
The right pulmonary artery was occluded after transplantation, making the dogs entirely dependent on the donor lung for gas exchange. In 3 recipients, the occlusion was performed immediately after the transplantation. In the other 3 dogs, the occlusion was performed 1 to 6 hours after the transplantation due to continued donor lung atelectesis. The dogs had maintainence anesthesia using 30 mg/kg of sodium pentobarbital. A Harvard volume-cycler respirator maintained the respiration at 10 to 20 respirations per minute after the operation, and tidal volume was maintained from 500 to 700 mL. A gas mixture of 50% O2, 3% CO2, and 47% N2 was used. A Gould pressure transducer was connected to the inspiration tubing for continuous measurement of inspiratory pressures for airway resistance calculations. Arterial blood samples were taken every hour for blood gas measurement, and venous blood samples were taken every 4 hours for hematologic, blood chemistry, and enzyme measurements. An intravenous drip of 5% glucose plus penicillin (1,000,000 units/L) and Flagyl (500 mg/L) was given to keep the blood glucose level at 80 to 120 mg/dL. The animal was sacrificed after 24 hours of observation, and lung tissues were examined for pathologic changes after transplantation.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). The maximum inspiratory pressure ranged from 13 ± 2 to 22 ± 3 mm Hg. Calculated airway resistance ranged from 0.012 ± 0.003 to 0.019 ± 0.004 mm Hg/mL. A slight increase in airway resistance occurred at 24 hours (Fig 2). Pulmonary systolic pressure ranged from 22 to 29 mm Hg during the preservation period and had a slight increase at the end of preservation.
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In two experiments, lung tissue samples were taken during the preservation period for electron microscopic studies. Representative photos are shown in Figure 3A and 3B; these samples were taken from the lung at 12 and 24 hours of preservation, respectively.
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). No arrhythmia occurred after the transplantation.
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). Arterial oxygen tension was maintained from 205 ± 39 to 360 ± 57 mm Hg, arterial carbon dioxide tension was maintained from 23 ± 2 to 34 ± 2 mm Hg, and arterial pH was maintained from 7.33 ± 0.04 to 7.51 ± 0.02 (Fig 6). When the transplantation procedure exceeded 1.5 hours, some areas of atelectasis were found, and frequent lung expansions were required. In one experiment, the recipient had a high fever and high white blood cell count before transplantation. Oxygen tensions were low after transplantation, and opposite pulmonary artery occlusion was also delayed. This dog died 8 hours earlier than the expected 24-hour observation. At autopsy, heart worms were discovered in the distal pulmonary arteries. Mean lung tissue wet/dry ratio obtained 24 hours after transplantation was 5.23 ± 0.23, which was greater than that of normal dogs (4.91 ± 0.10).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Although the heart-lung autoperfusion preparation is not suitable for clinical lung preservation, this technique is helpful in examining preservation techniques. Several factors can cause failure in lung preservation. The greatest limiting factor for the lung preservation is early gas exchange failure. Even though preservation for the heart has been successful for more than 12 hours in experimental settings, the lungs deteriorate quickly and usually become unusable after 4 to 6 hours of preservation [20]. Microthrombi and macrothrombi can occur early, even before harvest and preservation and become frank hemorrhagic blotches with reperfusion. Additionally, the lungs gradually stiffen, increase in weight, and end with severe lung damage.
Hibernation induction trigger may be useful when given to donors before organ harvest, as studies have shown that the lipoprotein membranes of erythrocytes from hibernating animals have increased levels of unsaturated fatty acids and demonstrate increased resistance in osmotic fragility tests, are more pliable, and are not susceptible to rouleaux formation in constricted capillary beds in hibernating animals [21]. Neither human cells nor ground squirrel (Citellus tridecemlineatus) erythrocytes agglutinate in ground squirrel serum at low temperatures, and ventricular blood drawing from hibernating woodchucks does not require heparinization of needles because the blood will not clot. In autologous transplants, kidneys obtained from hibernating ground squirrels were viable and suitable for transplantation for up to 10 days after harvesting, whereas kidneys from summer-active animals were successfully transplanted only at a maximum of 3 days [22].
In our original studies in which the dog multiorgan block was infused with physiologic saline solution, severe hepatic congestion occurred as early as 3.0 to 4.0 hours after preservation and reduced survival time of the multiorgan block to an average of 14.8 hours [14]. However, when plasma from deeply hibernating woodchucks was infused, survival time of the multiorgan autoperfusion system increased almost threefold to 44.6 hours [14]. A direct effect of infusion of HIT-containing plasma was the visually observable reduction of hepatic congestion. Within minutes of infusion, the liver, which had developed dark splotches over its entire surface and was hard to the touch, cleared and became supple and its appearance returned to normal. Comparable multiorgan survival times (averaging 46 hours) and the effects on the liver were achieved when the autoperfusion system was infused with the delta opioid DADLE [15], which mimics the natural biological activity in hibernators of the winter hibernating plasma [23].
Additionally, in our prior multiorgan preservation studies, we found that the inferior vena cava contained contrast particles, as seen by ultrasound imaging, that moved with the blood flow and appeared to increase in size and number during the course of preservation. Ultrasonic detection of large numbers of particles in perfusate during cardiac operations, as a result of cardiopulmonary bypass procedures, has previously been associated with multiorgan failure and a poor prognosis [24]. Serial lung sections showed clumps of white cells in the vascular space. These two findings suggest the possibility that one reason for progressive organ dysfunction during the autoperfusion studies might be an embolization by platelet and neutrophil aggregates in the heart, lungs, liver, and kidney. The effect of HIT on platelet aggregation was studied in 1 dog during the preservation protocol. Platelets behaved normally before HIT administration, with a dose-response relationship between the amount of adenosine diphosphate added and the extent of aggregation. However, after HIT infusion, even though the platelets aggregated normally in response to adenosine diphosphate, the platelets disaggregated shortly after adenosine diphosphate-stimulated aggregation, despite the use of high doses of adenosine diphosphate.
Efforts to identify the HIT molecule(s) have produced few results until recently. We have identified an 88-kDa plasma protein from woodchucks that is present only during hibernation. We have purified and concentrated this protein and have currently identified 39% of its amino acid sequence. In vitro assays using a chromatography fraction containing a high concentration of this 88-kDa fraction have shown it to be opiate-like in nature, its effects being reversible by opiate antagonists. We are presently cloning the gene for this protein to obtain complete identification and facilitate testing.
There are several reasons that make lung tissue preservation more difficult than for other organs: (1) The unique, delicate architecture of the lung poses a special problem in preservation. Methods effective for the short-term storage of kidneys, livers, and hearts do not necessarily work for lungs. (2) Functional dependence is placed on the preserved lung after it is transplanted. This makes evaluation of functional adequacy more critical during preservation.
The mechanism(s) by which HIT increased tissue survival time are postulated to be (1) reduced tissue metabolism, (2) reduced or eliminated platelet or leukocyte aggregation, and (3) improved microcirculation via vasodilation. This study clearly demonstrated the profound beneficial effects of HIT infusions on extending lung viability for successful transplantation using a multiorgan autoperfusion model. However, we are aware that the practical clinical utility of this model is somewhat limited, and that the donor lung atelectasis noted in some experiments was related to technical limits of this model rather than to difficulty with the use of the HIT-containing plasma infusions. We have now begun the use of HIT supplementation in standard ex vivo preservation, and it appears to markedly enhance preservation.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Supported in part by US Army Medical Research, Acquisition, Logistics and Development Command contract DAMD 17-92-C-2026 and VA medical research funds.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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