Ischemia-reperfusion injury of the spinal cord: Protective effect of the hydroxyl radical scavenger dimethylthiourea Willem Wisselink, MD, Samuel R. Money, MD, Donald E. Crockett , MD, Justin H. Nguyen, MD, Mark O. Becket, MD, Gist H . Farr, MD, and Larry H. Hollier, MD, New Orleans, La.
Purpose: This study was undertaken to evaluate whether neurologic outcome after aortic cross-clamping in rabbits could be improved with perioperative infusion of the hydroxyl radical scavenger dimethylthiourea and, if so, to determine whether it is effective during the period of ischemia, reperfusion, or both. Methods: In 41 New Zealand White rabbits, a snare occlusion device was placed at operation around the infrarenal aorta and tunneled into a subcutaneous position. Animals were then allowed to recover and, 48 hours later, randomized into four groups. In each group, the infrarenal aorta was occluded by tightening the snare in the awake animal. In groups 1, 2, and 3, cross-clamp time was 21 minutes. Group 1 (control) animals received saline solution, whereas group 2 (preclamp 21) received dimethylthiourea 750 mg/kg intravenously just before aortic damping. In group 3 (prerep 21), dimethylthiourea was given just before reperfusion. Group 4 received dimethylthiourea before damping, with cross-clamp time extended to 31 minutes. A second dose of saline solution or dimethylthiourea was given 12 hours after damping in controls and the three treatment groups, respectively. Animals were observed for 5 days, and final neurologic recovery was graded by an independent observer. Animals were then killed, and their spinal cords were removed for histologic examination. Results: Complete paraplegia and marked histologic spinal cord injury at 5 days were seen in 91% (10/11) of group i (control) animals, whereas all animals in group 2 (predamp 21) showed neurologic recovery (p < 0.0001). In group 3 (prerep 21), the final paraplegia rate was 50% (5 of 10), in group 4 (preclamp 31), 100% (10 of 10). Conclusions: Our results suggest that hydroxyl radicals play an important role in ischemia-reperfusion injury of the spinal cord and that treatment with dimethylthiourea can prevent paraplegia after 21 minutes of aortic cross-clamping in rabbits. (J VAsc SVRG 1994;20:444-50.)
Repair of thoracoabdominal aneurysms is com- plicated by paraplegia in up to 30% of patients, depending on the extent of aorta involved, the presence of dissection or rupture, hypotension, and cross-clamp time. 1-4 This complication has been attributed to permanent or transient ischemia of the
From the Ochsner Clinic, New Orleans. Presented at the Eighteenth Annual Meeting of the Southern
Association for Vascular Surgery, Scottsdale, Ariz., Jan. 26-29, 1994.
Reprint requests: Samuel R. Money, MD, Ochsner Clinic, Department of Surgery, 1514 Jefferson Highway, New Orleans, LA 70121.
spinal cord caused by interruption of blood flow during aortic clamping.
In most patients in whom postoperative paraple- gia develops, neurologic deficits are present at the time the patients awaken from anesthesia. However, in a subset of patients, the neurologic function is normal directly after the operation, only to deterio- rate during the ensuing days, starting up to 7 days after operation? Such patients are referred to as having delayed onset paraplegia. This phenomenon has led to speculation that at least part of the damage to the spinal cord occurs after blood flow has been restored, during the period of repeffusion. Oxygen- derived free radicals have been implicated as media- tors of reperfusion injury in various organ systems, including the central nervous system (CNS). s Con- sequently, treatment with appropriate scavengers can
JOURNAL OF VASCULAR SURGERY Volume 20, Number 3 Wisselink et al. 445
be expected to limit tissue damage. Popular agents such as superoxide dismutase and catalase have been disappointing as protectors of the CNS because of their inability to cross the blood-brain barrier, or, when concerning the spinal cord, the blood-nerve barrier. ~,6 Dimethylthiourea is a hydrogen radical scavenger that enters the CNS because of its lipid solubility and small molecular weight. It has been shown to reduce brain infarct size after middle cerebral artery occlusion in rats. 7 Previous studies in our laboratory showed a predictable pattern of neurologic deficits after infrarenal aortic cross- clamping in the rabbit. Further investigations have documented the sensitivity of this model to test the effect of therapeutic interventions that may protect the spinal cord. s,9
The primary aim of this study was to evaluate whether neurologic outcome after aortic clamping in rabbits could be improved with perioperative intravenous administration of dimethylthiourea. Second, we attempted to define whether free radi- cal-induced injury in the spinal cord is either restricted to the period of reperfusion (as generally believed), or also occurs during the time of ischemia.
MATERIAL AND METHODS
Forty-one New Zealand white rabbits (2 to 3 kg) were premedicated with atropine sulfate (0.005 mg/kg), administered intramuscularly, and anesthe- tized with intramuscular ketamine hydrochloride (40 mg/kg) and xylazine (10 mg/kg). Intermittent intra- venous readministration of one quarter doses of the anesthetic agents were given to maintain an adequate level of anesthesia and prevent the need for endotra- cheal intubation and mechanical ventilation. The fur on the left flank was clipped with electric shears, and the skin was prepared with iodine solution. With the animal positioned on the right side, a vertical flank incision of approximately 4 cm length was made to allow retroperitoneal dissection and exposure of the infrarenal aorta. A circumaortic occlusion device consisting of PE-50 tubing (Clay Adams, Parsip- pany, N.J.), a segment of 18F catheter and a button, was loosely placed just below the renal arteries. The tubing was tunneled into a subcutaneous pocket and secured for easy access later, and the animals were allowed to recover. Forty-eight hours later, two leads of a neurosensory stimulation unit were attached by alligator clips to the skin of the left hindlimb, and the animal was stimulated every 5 seconds with the lowest voltage required to assess motor and sensory function. The ends of the occlusion device were
retrieved by removing the stitch over the subcutane- ous pocket. Animals were randomized into four groups, and, in each group, infrarenal aortic occlu- sion was achieved by tightening the occlusion device, with the rabbit in an alert and awake state. In previous experiments s,9 in our laboratory we found that complete aortic occlusion, as recorded via implantable Doppler flow probes, can be reliably obtained when a consistent amount of tension is applied to the snare by one and the same surgeon who is blinded to the treatment protocol. In groups 1, 2, and 3, cross-clamp time was 21 minutes. Group 1 (control) animals received saline solution, whereas group 2 (preclamp 21) received dimethylthiourea 750 mg/kg, intravenously just before aortic clamp- ing. In group 3 (prerep 21), dimethylthiourea was given just before reperfusion. Group 4 received dimethylthiourea before clamping, with cross-clamp time extended to 31 minutes. A second dose of saline solution or dimethylthiourea was given 12 hours after damping in controls and the three treatment groups, respectively. Animals were observed for neurologic recovery over the following 96 hours and graded by an independent observer with use of a modified Tarlov-scale I° (grade 0, no movement of the lower limbs; grade 1, minimal movement; grade 2, good movement but unable to stand; grade 3, able to stand and walk but unable to hop normally; grade 4, normal recovery). Animals graded as 0, 1, or 2 were considered paraplegic, whereas grades 3 and 4 were grouped as normal. Final recovery was charac- terized as acute paraplegia, delayed onset paraplegia, or normal, determined by observed neurologic func- tion at two different time intervals: animals that were paraplegic at 5 hours after reperfusion were consid- ered acutely paraplegic, whereas those neurologically normal at 5 hours but paraplegic at 5 days were labeled as having delayed onset paraplegia. Five days after clamping, or at the time of acute permanent paraplegia, animals were overdosed with pentobar- bital (65 mg/kg), and the spinal cord was removed for fixation in formalin. Sections of the spinal cord were stained with hematoxylin-eosin and interpreted by a board-certified pathologist who was blinded to the treatment protocol. The abdominal aorta and large branches were examined for the presence of thrombosis. If present, the animal was excluded from statistical analysis. The research protocol has been approved by the Institutional Animal Care and Use Committee (approval number 9103).
All values are expressed as mean _+ standard error of the mean. Data were analyzed by unpaired Student's t test and Fischers' exact test. Statistical
JOURNAL OF VASCULAR SURGERY 446 Wisselink et al. September 1994
Table I. Neurologic function graded with the Tarlov scale 5 hours after aortic clamping
Group I Group iI Group l l I Group IV Tarlov Grade (Control) (Preclamp 21) (Prerep 21) (Preclamp 31)
0 2 0 1 9 I 1 0 1 1 II 0 0 1 0 III 3 1 3 0 IV 5 9 4 0
Tot~ 11 10 10 10
See text for description of groups.
significance was accepted when the value ofp was less than 0.05.
RESULTS
Occlusion of the infrarenal aorta in the awake rabbit caused complete, flaccid paraplegia within 28 + 13 seconds in all animals. The period of ischemia needed to cause loss of sensory fimction was significandylonger (240 + 68 seconds)(p < 0.05), without significant difference between groups. All animals lost control of their urinary sphincters during the period of aortic clamping. None of the animals showed physical signs of discomfort or pain. After restitution of aortic flow, 27% (3 of 11) of group 1 (control) animals stayed paraplegic (grades 0 to 2), whereas the remaining animals (8 of 11) showed return of motor function within 131 + 49 minutes of reperfusion time (grades 3 and 4) (Table I). However, in 87% of those control animals that recovered, neurologic function began to deteriorate, starting 12 to 24 hours after unclamping, and progressed to complete (delayed onset) paraplegia within the following 2 days. Although one animal remained neurologically normal (grade 4), the final paraplegia rate in controls was 91% (10 of 11) (Table II). Final paraplegia rate in group 2 (preclamp 21) was zero (0 of 10): motor function returned to grade 3 (n = 1) or 4 (n = 9) in all dimethylthiourea- treated animals within 120 + 50 minutes of reper- fusion and remained unchanged throughout the period of observation. The observed difference in final neurologic outcome between these two groups is statistically significant to a p value of less than 0.0001. Final neurologic outcome in group 3 (prerep 21) was intermediate: five of 10 animals were normal at 5 days and the remaining five had paraplegia, (three acute and two delayed onset). The observed differ- ence in neurologic function at 5 days between group 3 andgroup I was statistically significant (p < 0.05), whereas that between groups 3 and 2 approached significance (p = 0.051). In group 4 (preclamp 31),
all animals were paraplegic at both 5-hour and 5-day intervals.
At the time of sacrifice, none of the animals showed macroscopic or microscopic signs of throm- bosis in either the aorta, its major branches or the microcirculation in the spinal cord. Histologic ex- amination revealed destruction of anterior horn cells, nuclear disintegration, perikaryal swelling, and in- farction in the lumbar sections of the spinal cords of all paraplegic animals, and the result was normal in those who had fully recovered (Fig. 1).
DISCUSSION
The basic mechanisms of neurologic injury after aortic clamping have not been completely uncovered, and methods to prevent it, including improved technique, anaesthetic management, shunts, pharma- cotherapy, and spinal fluid drainage, have been at best partially effectiveJ ,n During the period of aortic cross-clamping, blood flow to the intestines, liver, kidneys, and spinal cord is diminished or completely interrupted, resulting in various degrees of tissue ischemia. Clearly, longstanding ischemia during aor- tic clamping and failure to restore flow to the spinal cord will lead to spinal infarction and irreversible neurologic deficits. In addition, recent evidence has suggested that a substantial component of the injury is not due to the ischemia per se but, instead, takes place after the ischemic interval, during the period of reperfusion. 12 A multitude of factors have been suggested to play a role in reperfusion injury of the spinal cord. Postperfusion hyperemia may lead to simple physical changes that result in swelling and edema of the cord. Edema within the confines of the subarachnoid space may cause compromise of venous and later arterial flow. This concept is supported by the beneficial effects of spinal fluid drainage. 13 B arone and colleagues 14 found a strong relation between the severity of post-ischemic spinal cord hyperemia and the incidence of paraplegia in dogs. Robertson and Grossman is noted that lactic acid levels in the rabbit
JOURNAL OF VASCULAR SURGERY Volume 20, Number 3 Wisselink et al. 447
Table II. Neurologic function graded with the Tarlov scale 120 hours after aortic clamping
Group I Group 1I Group 11I Group IV Tarlov Grade (Control) (Preclamp 21) (Prerep 21) (Preclamp 31)
0 8 0 3 10 I 2 0 2 0 II 0 0 0 0 III 0 1 1 0 IV 1 9 4 0
Total 11 10 10 10
spinal cord were higher during the early period of reperfusion than during ischemia. These authors speculate that the aerobic metabolism requires some time to recover after a period ofischemia and that the lactic acidosis during reperfusion is caused by the increased availability of glucose. However, there is no evidence that this "reperfusion acidosis" is directly related to neurologic outcome. Other suggested causative factors in reperfusion injury include vaso- spasm from release of thromboxane or leukotrienes and cytotoxic destruction by leukocytes and macro- phages. 16'17 Most popularly, however, reperfusion injury is believed to be specifically related to the sudden availability of excess quantifies of molecular oxygen in ischemic tissue. Since the seminal work of McCord 18 and Bulkley 19 in the early 1980s, it has been accepted that reactive oxidant species, including the superoxide anion, hydrogen peroxide, and the hydroxyl anion, play an important role in the development of ischemia-reperfusion injury in vari- ous organs. The xanthine oxidase system has been shown to be the primary source of these oxygen- derived free radicals in the setting of ischemia followed by reperfusion. Initial products are the superoxide anion and hydrogen peroxide. These compounds may combine in a trace metal-catalyzed reaction to form the hydroxyl radical, which is more reactive than either of its precursors and causes tissue damage by inducing lipid peroxidation. 2°
Recent studies in the rat, cat, and dog 2~-23 have suggested that oxygen-derived free radicals are also involved in ischemia-reperfusion injury of the brain. Tissue damage was diminished in these studies by blocking the pathways by which oxygen radicals are produced or by indirect measurement of superoxide anion radicals in the cerebral extracellular space. As stated, subsequent attempts to ameliorate brain or spinal cord ischemic injury with radical scavengers have generally been disappointing in part because most of the tested agents do not cross the blood brain barrier. Dimethylthiourea is a hydroxyl radical scav- enger that has been shown to rapidly enter the central
nervous system as a result of its lipid solubility and small molecular weight. 7 In vitro studies by Jackson et al.~ demonstrated decreased concentrations of hy- drogen peroxide and hydroxyl radical after addition of dimethylthiourea to a mixture containing human neutrophils, as well as inhibition of oxygen metabolite-dependent neutrophil killer function. The latter observation provides us with a possible addi- tional beneficial effect of dimethylthiourea in spinal cord ischemia: neutrophils have been widely impli- cated in the pathogenesis of reperfusion injury. 16 Martz and colleagues 7 reduced cerebral infarction af- ter middle cerebral artery occlusion in rats with the use of intravenous dimethylthiourea. Our finding that dimethylthiourea, when given before cross- clamping, completely prevented paraplegia after 21 minutes of ischemia suggests that hydroxyl radicals are a major factor in the pathogenesis of ischemia- reperfusion injury of the spinal cord. As outlined above, although the involvement of oxygen-derived free radicals has been widely implicated in the patho- genesis of reperfusion injury, less thought has been given to potential deleterious effects during the pe- riod of ischemia. In our study, animals that were given dimethylthiourea shortly before restitution of aortic flow, and thus were denied a possible therapeu- tic effect during the period ofischemia, fared less well than those who received the drug before aortic clamping, suggesting that, indeed, oxygen radicals cause injury during the ischemic period.
The protective effect of dimethylthiourea during the period of ischemia, however, is limited, as demonstrated by the poor results in group 4 (pre- clamp 31). These animals apparently sustained irre- versible spinal cord injury during 31 minutes of ischemia, precluding a potential therapeutic effect of dimethylthiourea during the period of reperfusion. This finding is compatible with the work of Parks and Granger, 2s who found that beyond a certain degree of ischemic injury to rat small intestine, manipulation of the free radical mechanism could not improve final outcome after reperfusion.
JOURNAL OF VASCULAR SURGERY 448 Wisselink et aL September 1994
Fig. 1. Histologic appearance of sections of lumbar spinal cord. (Original magnification × 20,) Stained with hematoxylin-eosin. Above: dimethylthiourea-treated animal (group 1), normal appearance. Below: paraplegic animal, destruction of anterior motor horn cells, nuclear disintegration, perikaryal swelling, and infarction.
Most of the control animals in our study initially showed full neurologic recovery. Similar to what is seen in human beings after thoracic aortic clamping, a number of animals subsequently began to deterio- rate and progressed to complete (delayed onset) paraplegia in several days. The fact that treatment with dimethylthiourea just before reperfusion could reduce this number and did not affect the incidence of acute paraplegia, suggests that delayed onset paraplegia is primarily a result of reperfusion injury to the spinal cord. In an earlier study in our laboratory, 26 the incidence of delayed onset paraplegia was reduced by gradually reperfusing the ischemic spinal cord, leading to the same conclusion.
There are several limitations to our ability to draw conclusions from this study. Factors that may influ- ence neurologic outcome, such as body temperature, hematocrit, blood gases, blood glucose concentra- tion, or spinal fluid pressure, were not measured. The necessary placement of arterial cannulas, temperature
probes, or spinal catheters cannot be reasonably performed in the awake, unsedated animal and could by itself influence neurologic outcome. The exact mechanism by which dimethylthiourea affects neu- rologic outcome after aortic clamping is not clear after this study, and our speculation that hydroxyl radicals are involved needs further proof.
We did, however, show that paraplegia after a limited period of aortic clamping in rabbits can be prevented with dimethylthiourea if infusion is started before the ischemic period. The findings in this study further suggest that free radicals are active during both the periods of ischemia and reperfusion. The incidence of delayed onset paraplegia in particular seems to be related to events that occur during the period of reperfusion.
We gratefully acknowledge the expert assistance of Mr. Michael A. Chambers, Mrs. Donna B. Scarbrough; and Mrs. E. Luxufie Sorenson.
JOURNAL OF VASCULAR SURGERY Volume 20, Number 3 Wisselink et al. 449
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3. Hollier LH, Money SR, Naslund TC, et al. Risk of spinal cord disfunction in patients undergoing thoracoabdominal aortic replacement. Am J Surg 1992;164:210-4.
5. Kirshner DI, Kirshner RI, Heggeness LM, Deweese JA. Spinal cord ischemia; an evaluation of pharmacologic agents in minimizing paraplegia after aortic occlusion. J VAsc SuR~ 1989;9:305-8.
6. Lira KH, ConnoUy M, Rose D, et al. Prevention of reper- fusion injury of the spinal cord: use of recombinant super- oxide dismutase. Ann Thorac Surg 1986;42:282-6.
8. Moore WM, Hollier LH. The influence of severity of spinal cord ischemia in the etiology of delayed onset paraplegia. Ann Surg 1991;213:427-32.
9. Naslund TC, Hollier LH, Money SR, et al. Protecting the ischemic spinal cord during aortic clamping: the influence of anesthetics and hypothermia. Ann Surg 1992;215:409-16.
10. Tarlov IM. Spinal cord compression: mechanism of paralysis and treatment. Springfield, Ill.: Charles C. Thomas, 1957: 147.
11. Wisselink W, Becker MO, Nguyen JH, Money SR, Hollier LH. Protecting the ischemic spinal cord during aortic clamping: the influence of selective hypothermia and spinal cord perfusion pressure. J VAsc SUWG 1994;19:788-96.
12. Cuevas P, Reimers D, CarceUer F, limenez A. Ischemic reperfusion injury in rabbit spinal cord: protective effect of superoxide dismutase on neurological recovery and spinal infarction. Acta Anat 1990;137:303-10.
13. Bower TC, Murray MJ, Gloviczki P, Yaksh TL, Hollier LH, Pairolero PC. Effects of thoracic aortic occlusion and cere- brospinal fluid drainage on regional spinal cord blood flow in
dogs: correlation with neurologic outcome. J VASC SURG 1988;9:135-44,
14. Barone GW, Joob AW, Flanagan TL, Dunn CE, Kron IL. The effect of hyperemia on spinal cord function after temporary thoracic aortic occlusion. J VAsc SURG 1988;8:535-40.
15. Robertson CS, Grossman RG. Protection against spinal cord ischemia with insulin-induced hypoglycemia. J Neurosurg 1987;67:739-44.
16. Schurer L, Grogaard B, Gerdin B, Arfors KE. Effects of neutrophil depletion and superoxide dismutase on postisch- emic hypoperfusion of rat brain. Adv Neuro11990;52: 57-62.
17. Clark WM, Madden KP, Rothlein R, Zivin JA. Reduction of central nervous system ischemic injury in rabbits using leukocyte adhesion antibody treatment. Stroke 1991;22:877- 83.
18. McCord JM. The superoxide free radical: its biochemistry and pathophysiology. Surgery 1983;94:412-4.
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Submitted Feb. 3, 1994; accepted April 19, 1994.
DISCUSSION
Dr. Francis Robicsek (Charlotte, N.C.). In our ongoing work using immunohistologic methods during the time of ischemia, parallel with ventricular function, we found what most practicing surgeons already believe: Tissue damage occurs during ischemia, but it manifests after the blood flow is restored. Reperfusion, especially done improperly, can do harm.
Urea products have been known for a long time to protect brain tissue from ischemia. Thus the phenomenon demonstrated by the authors may be due to mitigated effects of ischemic damage rather than prevention of
reperfusion damage. That this is the case is indicated when the authors extended ischemia time to 31 minutes. Regardless what they did before reperfusion in the rabbits, it did not help. Also, when the urea was given before reperfusion but after ischemia, it did not provide more protection than you can get with pentobarbital anesthetic itself.
Dr. Brent T. Allen (St. Louis, Mo.). You have presented impressive results using a free radical scavenger to reduce ischemic spinal cord injury. Another major factor in the pathogenesis of ischemic spinal cord injury is the
• JOURNAL OF VASCULAR SURGERY 450 Wisselink et al. September 1994
liberation of potentially toxic amino acid neurotransmitters such as glutamate and aspartate. I wonder if this drug could have an effect on this pathway, apart from being a free radical scavenger. We have measured spinal cord adenosine triphosphate (ATP), lactate, glutamate, and aspartate concentrations before, during, and after spinal cord isch- emia in the rabbit. ATP concentrations are similar in animals that are protected from paraplegia with hypother- mia when compared with normothermic controls that become paraplegic. Because the products of ATP degrada- tion are a major substrate for free radical generation on reperfusion, both hypothermic and normothermic animals should have a similar potential for free radical production.
We noted that the best predictors of paraplegia were the lactate, aspartate, and glutamate concentrations after reperfusion. In paraplegic animals the intracellular concen- trations of two amino acid neurotransmitters decrease, whereas lactate levels remain elevated on reperfusion. In neurologically normal animals, the lactate concentration normalizes on reperfusion, and the glutamate and aspartate levels remain normal or increase slightly. I wonder whether dimethylthiourea has some effect on lactate metabolism or on these potentially toxic amino acid neurotransmitters.
Dr. G. Melville Williams (Baltimore, Md.). Two concepts of reperfusion injury are emerging. One that is mediated through the superoxide/hydroxyl radical and the other occurring in the central nervous system related to a phenomenon called "excitotoxicity," where basically the amino acid neurotransmitters accumulate during periods of ischemia. When the receptors become expressed after reperfusion, you end up with a tremendous mess of overexcitement, basically stimulating the neurons to death. This can occur both acutely and in delayed fashion in experimental models, and it seems to me that this second
mechanism of excitocytotoxicity is becoming more and more interesting. Phenomenon that seem inexplicable, like the value of naloxone, may well have its foundation in the blockade of this second mechanism.
Dr. Willem Wisselink. We found that the neurologic function at 5 hours significantly improved with just one dose of dimethylthiourea, but that a certain number of animals subsequently went on to develop delayed onset paraplegia. The latter could be prevented with a second dose of dimethylthiourea, 12 hours after clamping.
I think we agree that the major damage is done during the period of ischemia, but this does not necessarily mean that one cannot improve the clinical end result by therapeutic intervention during the period of reperfusion.
I agree that this experimental model has some serious shortcomings. With rabbits awake and alert during cross- clamping, it is very difficult to do additional monitoring or measurements of compounds like lactate, ATP, and exci- tatory amines. Neurologic fianction, however, can be compared accurately, and screening studies with this rabbit model should be followed with a more detailed evaluation of promising compounds and interventions. Also, the critique that we did not prove that the effect of dimethyl- thiourea on neurologic outcome is the result of its radical scavenging properties is valid. However, dimethylthiourea has been shown in studies in rat brain to specifically decrease the concentration of hydroxyl radicals and is not known to have other effects that have been implicated in ischemia-reperfusion injury of the spinal cord.
Obviously further work is needed to pinpoint the exact mechanism by which dimethylthiourea brings about its apparent beneficial effect on spinal cord fianction following aortic clamping.
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