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J Thorac Cardiovasc Surg 1995;109:1097-1102
© 1995 Mosby, Inc.
CARDIAC AND PULMONARY REPLACEMENT |
Loma Linda, Calif.
Address for reprints: Steven R. Gundry, MD, Professor and Head, Divisionof Cardiothoracic Surgery, Department of Surgery, Loma Linda University MedicalCenter, 11234 Anderson St., Loma Linda, CA 92354.
Abstract
A paucity of donor organs is the principal limitationin human heart transplantation. Prompted by our short-term studies of reanimating"dead" donor hearts in sheep, we applied the same reperfusion modificationsin juvenile baboons to determine human applications in an anoxic arrest model(as occurs when non-brain-dead patients are extubated and allowed to die).Ten juvenile baboons (mean weight 3.6 kg) were studied. Five baboons wereused as donors. After being anesthetized, donors were pretreated with methylprednisolone(Solu-Medrol), 50% dextrose, nifedipine, and prostaglandin E1 and then paralyzed and extubated. Donors becamepulseless at 7 ± 1 minutes and had electric arrest 9 to 18 minutesafter paralysis. The five donors were left undisturbed and warm for 15, 22,30, 30, and 31 minutes, respectively, after asystole. They were then given250 ml of 4° C Roe's crystalloid cardioplegic solution via the aorticroot and the hearts were explanted into iced Euro-Collins solution. Five baboonsserved as recipients. After donor harvest, recipients were placed on cardiopulmonarybypass, given prostaglandin E1 , and cooled to 18° C; circulatory arrest was instituted and the recipient'sheart excised. The donor heart was transplanted in an orthotopic position.Before reinstitution of bypass, 250 ml of terminal leukocyte-depleted bloodcardioplegic solution was given, then bypass was restarted and the heartswere reperfused for 60 minutes. All animals were weaned from bypass withoutthe use of inotropic agents. All animals were extubated within 2 to 4 hoursafter bypass and received standard immunosuppression. Peak creatine kinaseMB/total creatine kinase ratio was 0.2% ± 0.2%. Postoperative ejectionfractions by echocardiography were 75% to 80% (mean 76%). Animals survived1, 9, 13, 16, and 34 days, with three deaths caused by acute rejection andone each by stroke and diarrhea/dehydration. Pathologic findings showed noareas of fibrosis or ischemic damage. We conclude that successful reanimationand engraftment can be achieved with the use of the asystolic primate heart;this work suggests that human application is realistic and could greatly expand thedonor pool. (J THORAC CARDIOVASC SURG 1995;109:1097-102)
The lack of available donors continues to thwart the further applicationof cardiac transplantation. Efforts to educate the public have failed toproduce any increase in the number of transplants performed. Although mechanicalreplacement of failing human hearts shows promise, the use of human heartdonors clearly is preferable to any other alternative. Prompted by our successin using pediatric donor hearts that had periods of arrest and cardiopulmonaryresuscitation for up to 2 hours, we began a laboratory investigation intothe feasibility of "reanimating" dead pulseless donor hearts in lambs.
1 Lamb hearts were successfully usedfor orthotopic heart transplantation after 1/2 hour of cardiac electric standstillcaused by either anoxia or exsanguination. 2,3 Because species tolerance to ischemiavaries widely, before application of cardiac reanimation in human beings couldbe recommended, duplication of these results in a long-term subhuman primatemodel appeared mandatory.
In this study we investigated the application of simple reperfusionmodifications in a baboon heart transplant model that used "dead" hearts harvestedfrom baboon donors who had had an anoxic death, similar to the death thatoccurs when patients are extubated and allowed to die a compassionate deathwhen not fully brain dead.
MATERIAL AND METHODS
Ten juvenile Papio baboons of 2.9 to4.1 kg (mean 3.6 kg) were selected as random pairs; in cases in which a sizediscrepancy existed, the larger of the two animals was chosen as the donor.Thus five baboons were donors and five were recipients. The donor baboonswere anesthetized with ketamine hydrochloride, intubated, connected to a volumeventilator (Servo C, Siemens Corp., Lund, Sweden), and maintained with intravenouspentobarbital sodium. Eighteen-gauge intravascular catheters were placed inthe femoral vessels and the arterial pressure transduced. No predonation functionalstudies were done. The electrocardiogram was continuously monitored. All animalswere given 125 mg of intravenous methylprednisolone and 10 ml of intravenous50% dextrose and started receiving intravenous prostaglandin E1at 0.1 µg/kg per minute. A 5 mg capsule of nifedipine was opened and itscontents administered sublingually. Finally, 4 mg/kg of sodium heparin wasadministered. After 5 minutes of pretreatment, the donors were given 2 mgof pancuronium bromide (Pavulon) and the ventilator was disconnected.
Pulselessness occurred at 7 ± 1 minutes after paralysis; baboonshad electric arrest 9 to 18 minutes after paralysis Animals were left undisturbedfor 15, 22, 30, 30, and 31 minutes, respectively, and then a median sternotomywas done. An 18-gauge catheter was inserted into the ascending aorta, a bloodsample was collected for determination of arterial blood gas values at thetime of harvest, and the aorta was crossclamped. Two hundred fifty millilitersof Roe's crystalloid cardioplegic solution was instilled at 4° C intothe aorta, with simultaneous division of both venae cavae. The heart was excisedand placed for a mean of 1 hour in 4° C Euro-Collins solution to which5 ml of 50% dextrose had been added.
After donor harvest, recipient baboons were similarly anesthetized,the lungs ventilated, and maintenance with halothane inhalation instituted. A median sternotomy was done, and the pericardium was incised and fashionedinto a cradle. The animals were given 4 mg/kg of sodium heparin. The ascendingaorta was cannulated through a purse-string suture for arterial inflow andthe right atrial appendage cannulated for venous egress to the pump oxygenator.Animals were placed on cardiopulmonary bypass support with use of an asanguineouspriming solution and cooled to a rectal temperature of 20° C.
Once cardiopulmonary bypass was begun, administration of prostaglandinE1 was started at 01 µg/kg per minute and continued until 30minutes before gradual removal from bypass. At the same time, a 5 mg nifedipinecapsule was opened and its contents administered sublingually. After coolingwas completed, the ascending aorta was crossclamped and profound hypothermicarrest was instituted. Cardiac transplantation was done in our usual fashionwith the "dead" donor heart after the recipient's heart was excised. 4 The venous cannula was reinsertedinto the donor's right atrium. Through a separate aortic purse-string suture,an 18-gauge cannula was inserted into the ascending aorta. The left atrialappendage was vented, and the baboon was given 250 ml of leukocyte-depletedterminal blood cardioplegic solution into the aortic root. The baboon wasplaced back on bypass and rewarmed. The hematocrit value of the animal wasconcentrated up to 0.30 with an in-line ultrafilter circuit and calcium repletedduring a further 50-minute rewarming period. All five hearts returned to sinusrhythm before removal of bypass. No inotropic support was given.
After the termination of bypass and reversal of heparin with protaminesulfate, all baboons' chests were closed without drainage catheters. Allanimals were extubated within 4 hours of the operation and begun on oral feedingsthe first postoperative day. Blood was drawn before and after bypass and dailyfor 3 days for determination of creatine kinase (CK) and CK-MB fractions.Transthoracic echocardiograms were obtained every other day. Cyclosporinewas given orally at 30 to 50 mg/kg daily, azathioprine was administered orallyat 3 mg/kg daily, and 125 mg methyl prednisolone (Solu-Medrol) was administeredintravenously on the first postoperative day. Rejection episodes were treatedwith intravenous steroid boluses. When death occurred, autopsies were doneto determine the cause of death and hearts were examined microscopically forevidence of ischemic damage and rejection.
RESULTS
Donor hearts were harvested 15, 22, 30, 30, and 31 minutes after electriccardiac arrest. All hearts regained sinus rhythm and all hearts were weanedfrom cardiopulmonary bypass without inotropic support. All animals were extubatedfrom 2 to 4 hours after operation. Transthoracic echocardiograms demonstratedejection fractions from 75% to 80% (mean 76%). The mean peak CK-MB/total CKratio was 0.2 ± 0.2, with no heart eluting a large amount of myocardialCK, thus confirming the lack of significant ischemic damage.
Animals survived 1, 9, 13, 16, and 34 days, with the death on day 1caused by a stroke and the death on day 9 by dehydration. All other deathswere due to rejection. Autopsy on explanted hearts demonstrated minimal tono ischemic damage (Figs. 1 and 2), whereas rejectinghearts showed typical changes of grade 3 to 4 rejection.
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DISCUSSION
It has been assumed that anoxically arrested hearts would be poor donorsfor cardiac transplantation, in that the energy stores of the heart wouldbe severely depleted and that on reperfusion, contraction band necrosis wouldinvariably occur. 5 Cooper 6 and Lundsgaard-Hansen and associates, 7 working independently, had poorresults with such an animal model. However, since 1985 our clinical successwith the use of infant and pediatric donor hearts that had been successfullyresuscitated after arrest times of up to 1 hour prompted us to study how reperfusionmodifications might enable "dead" hearts to be reanimated and used immediatelyas donor organs. 1,8,9
Short-term experiments in lambs with both anoxic and exsanguinationmodels confirmed that reperfusion modifications, with or without donor pretreatment,could reliably and consistently produce "normal" donor hearts even after 30minutes of warm ischemia. Pretreatment of the recipient with sublingual nifedipine;prostaglandin E1 infusion; and reperfusion with a low hematocritvalue (3% to 5%), low ionized calcium (0.3 to 0.5 mg/dl), low pressure (meanarterial pressure 20 mm Hg), and low flow (25 ml/m2) blood reperfusatewas successful in producing short-term successful orthotopic transplantationand reanimation of these "dead" hearts. 2,3 However, because of species differencesand the lack of long-term results, direct applicability to human beings couldnot be assured.
In this experiment, as in the anoxic lamb model, donors were pretreatedwith steroids, prostaglandin, nifedipine, and dextrose, and recipients receivedthe same reperfusion modifications as noted for the lamb model. Additionally,a leukocyte-depleting filter was added to the terminal blood cardioplegicsolution, in large part because of the success of this intervention in heartspreserved for greater than 24 hours. 10
The lack of a significant reperfusion injury or permanent ischemic damageis attested to by the lack of CK-MB band elaboration, the lack of acute orchronic fibrotic/ischemic changes in the myocardium on postmortem examination,and the normal ejection fractions. These findings echo those seen in ourpatient population, in which neither short- nor long-term changes of ischemicdamage have been seen in transplanted but previously arrested hearts. 1,8,9 Although long-term survivalin our series was not achieved, with three animals dying as a result of rejection2 to 5 weeks after operation, we did not use high cyclosporine levels (range180 to 266 mg/dl by monoclonal antibody) or FK 506, a drug that we have previouslyshown produces superb long-term survival in baboons, even those receivingtransplant with xenografts. 11 Because we have not seen accelerated rejection in hearts with longischemic times, or in previously resuscitated hearts, 9,12 we doubtthat the deaths caused by rejection in this laboratory series can be attributedto ischemic injury.
In conclusion, Baboon hearts recovered 15 to 31 minutes after cardiacstandstill caused by anoxia can be transplanted, reperfused, and reanimatedwithout difficulty with the use of clinically applicable methods. These datasuggest that use of these techniques in human beings is feasible. If applied,the use of donors with nonbeating hearts could be extended to cardiac transplantation,thus greatly expanding the donor pool.
Appendix: DISCUSSION
Dr. Adnan Cobanoglu (Portland, Ore.).
During the past 5 years Dr. Gundry and his colleagues at Loma Linda
have given excellent reports on marginal resuscitated pediatric donors and
reanimated donors in juvenile lamb models, and this study is a natural extension
of their prior work.
There has been a steady increase in the number of patients waiting for transplantation. As of May 1994 there were 2900 patients on the waiting list for heart transplantation in the United States. Only 1900 to 2000 transplants are done a year and the attrition rate while waiting may be as high as 40% on some of the lists. In an attempt to meet an ever-increasing demand for organs, most programs have extended the criteria for graft acceptability. The upper limit of the acceptable donor age was the first area that was liberalized. The second area has been acceptance of the so-called marginal donors. These are donors who do not fit the hemodynamic criteria for acceptance, those who have had prolonged cardiopulmonary resuscitation, or those receiving large doses of inotropic drugs or who have compromised left ventricular function.
Our experience in Portland with 250 transplants and our recent review for the Society of Thoracic Surgeons program have shown that with careful preoperative and intraoperative management of the donor heart a significant number of these marginal hearts, even those with up to 30 to 40 minutes of cardiac massage and resuscitation and those initially receiving 20 to 30 µg of dopamine or the equivalent, can be salvaged and successfully used for transplantation.
Dr. Gundry's report takes this issue one step further. He has demonstrated that at least in the laboratory setting and in a subhuman primate, the juvenile baboon, successful heart transplantation is possible from not only brain-dead but also heart-dead donors. These are donor hearts with warm asystolic ischemia. This study is analogous to the discontinuation of ventilatory support for compassionate reasons in persons in vegetative states or in those who are hopelessly ill but are potential organ donors in whom a cardiac arrest and asystole will occur after the ventilator support is stopped.
I congratulate the authors for exploring this frontier, and I have a few questions Studies have shown different responses to ischemia or anoxia, or both, in mature versus immature myocardium. Is your study clinically applicable to all donors or just to the younger ones, that is, neonates and young infants?
Dr. Gundry.
These baboons are juveniles, that is, they are around 9 months to a
year old and in a baboon lifetime this is equivalent to a teenager. Our studies
at Loma Linda involving clinical donors have shown no difference in the tolerance
of adult hearts and infant hearts to ischemia, so I would like to think that
we could extrapolate these findings in a teenage model to a fully adult model.
Dr. Cobanoglu.
Did you evaluate the amount of myocardial injury by means other than
the CK-MB fraction such as myosin light chain efflux or other biochemical
parameters?
Dr. Gundry.
No. We looked at myosin light chain efflux in a clinical study that
Dr. Kawauchi in our laboratory did and found that the light chain efflux correlated
very well to the CK-MB band and also to late myocardial fibrosis. In following
the CK-MB band, plus the echocardiography and the lack of inotropic support,
we are fairly comfortable that no major ischemic damage was done.
Dr. Cobanoglu.
The duration of total ischemic time will be another important factor
in the clinical setting. Will this method be applicable in the clinical setting
where 3 or more hours of ischemia is common?
Dr. Gundry.
That is an area of new investigation. We wanted to mimic a clinical
setting in which a patient could be extubated in the intensive care unit even
with the family present and allowed to die. Then the patient would simply
be heparinized in that state and the surgical team either in the intensive
care unit or available in a room next door would explant the heart. I think
it is practical to obtain these hearts in 1/2 hour. It remains to be seen
whether we can take someone from outside the hospital who has been dead for
several hours and do the same manipulations.
Dr. Cobanoglu.
When do you plan to take the next step from subhuman primates to human
patients? Do you plan to use these reanimated hearts in high-risk recipients
or on all comers, including status II patients?
Dr. Gundry.
We are studying that question with our institutional review board right
now. There is an ethical question and also a legal question as to the definition
of death. If the definition of death is the irreversible cessation of heartbeat
(which is the common definition of death in most states), does that apply
to irreversible cessation of heartbeat in that particular person or does it
apply to when that heart is removed, implanted in someone else, and then started
again? Is that the same thing? Our legal team has advised us that the potential
exists that we could be charged with murder by doing this particular procedure
without legislative change in the law of defining death, so we are in limbo
at this point. Obviously donors with nonbeating hearts are used in the kidney
and liver fields, but again surgeons are not required to use that heart, which
still remains dead in that patient.
Dr. Cobanoglu.
Other than the murder charge, I think there is somewhat of an ethical
decision here, because the donor heart is really dead when you accept it.
How will you inform the patient? Will you tell him or her that you have a
"good dead heart" available?
Dr. Gundry.
I think many of our patients who are in extremis would be more than
happy to take a dead heart from somebody else, particularly given the evidence
that these hearts are resuscitatable. We certainly use older hearts on older
recipients now, something that was unthinkable several years ago, and I can
assure you that my older recipients are perfectly happy to have an older heart
as long as they are alive. I think this is just one more step in a direction
that we have to go as soon as the legal ramifications are worked out, and
I am sure they can be. The point of this paper is this is the next step and
it can be done.
Dr. Dimitri Novitzky (Tampa, Fla.).
This is a nice animal model, but I think clinical implementation is
very unlikely. In Cape Town we induced experimental brain death in baboons
and documented a massive autonomic response in which there is a significant
increment of circulating and endogenous catecholamine release that induces
diffuse myocardial injury. There is marked subendocardial injury, as well
as scattered myocyte necrosis. These observations occur as early as 30 minutes
after induction of brain death. Pretreatment with calcium blockers such as
verapamil abolished the catecholamine-induced injury without observation of
histologic myocyte necrosis.
I would like to ask you how the initial neurologic (sympathetic) impact observed in our animal model and human donor hearts will be prevented? In this particular experimental model, after anesthesia and calcium blocker pretreatment, anoxic brain death is induced, which therefore avoids catecholamine-induced injury.
In a clinical setting, I find it hard to believe this can be applied The brain-dead organ donors, during the agonal period, already have been exposed to the catecholamine-induced injury. The slide you showed us had a normal histologic subendocardium similar to the experiments done by us.
Dr. Gundry.
I agree with you that from some experimental studies this is true.
However, in all of our laboratory studies with this identical model we have
yet to produce that subendocardial injury. I think it is secondary to the
modifications of how reperfusion is done. Of course, the big fear with the
use of marginal donors was that these donors had huge catecholamine storms
and had marginal myocardial conditions with a very low ejection fraction by
echocardiography and huge amounts of inotropic support. The fear was that
these were severely damaged hearts that would not work in human subjects.
In fact, as Dr. Cobanoglu has pointed out and in our study, this simply does
not happen when these hearts are transplanted. When they are taken out of
the deleterious milieu of the brain-dead donor and put in a normal human being,
these hearts respond normally, and their inotropic support is minimal. Even
patients whose donor hearts have received norepinephrine, epinephrine, and
huge amounts of dopamine are weaned from cardiopulmonary bypass with minimal
inotropic support in the normal milieu of a healthy brain, if you will.
Additionally, Dr. Javier Alonzo de Begona, at our institution, has looked at the amount of fibrosis in hearts recovered from children who have had as long as 1 hour of downtime before cardiopulmonary resuscitation, a study that now extends to 8 years after transplantation, and has found no difference in the amount of myocardial fibrosis or in the ejection fraction of these hearts in human beings compared with these findings in hearts from donors who have not had cardiopulmonary resuscitation and have not had downtime. I suspect that in fact these laboratory data are directly transferable to the human subject.
Footnotes
From the Departments of Surgerya
and Surgical Pathology,b Loma Linda University Medical Center,
Loma Linda, Calif. 
Read at the Twentieth
Annual Meeting of The Western Thoracic Surgical Association, Olympic Valley,
Calif., June 22-25, 1994.
References
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