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Ann Thorac Surg 2002;74:846-850
© 2002 The Society of Thoracic Surgeons
a Division of Vascular and Endovascular Surgery, University of Tennessee Medical Center, Knoxville, Tennessee, USA
b Division of Thoracic and Cardiovascular Surgery, Departments of Surgery and Molecular Pharmacology, University of Virginia Health System, and Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA
* Address reprint requests to Dr Kern, Department of Surgery, Box 181-95, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
e-mail: jak3r{at}virginia.edu
Presented at the Forty-eighth Annual Meeting of the Southern Thoracic Surgical Association, San Antonio, TX, Nov 8–10, 2001.
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Abstract |
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Methods. Eighteen rabbits underwent laparotomy, and 14 sustained spinal cord ischemia from cross-clamping the infrarenal aorta for 45 minutes. One group (ischemia-reperfusion [I/R] + ATL) received ATL-146e intravenously for 3 hours during spinal cord reperfusion. A second group (I/R) received equivolume intravenous saline solution for 3 hours and served as an ischemic control, and a third group (Sham) underwent sham laparotomy. At 48 hours, all subjects were assessed for motor impairment using the Tarlov scoring system (0 to 5). Lumbar spinal cord sections were immunolabeled for A2A-R and graded in a blinded fashion using light microscopy.
Results. There was a significant improvement in Tarlov scores in I/R + ATL animals compared with the I/R group. Sham-operated animals demonstrated no A2A-R immunoreactivity. There was a dramatic increase in A2A-R immunoreactivity in neurons of lumbar spinal cord sections from I/R compared with I/R + ATL and sham-operated animals.
Conclusions. Reduction in paralysis in animals receiving ATL-146e correlates with the new finding of A2A-R expression on lumbar spinal cord motor neurons after ischemia. Adenosine A2A agonists may exert neuroprotective effects by binding to inducible neuronal A2A-R that are upregulated during spinal cord reperfusion, and reduced in response to administration of an A2A-R-specific agonist.
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Introduction |
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Microscopic evaluation of the lumbar spinal cord revealed that there is a preservation of neuronal viability after spinal cord ischemia-reperfusion (I/R) in rabbits treated with ATL-146e compared with ischemic controls [3]. Spinal cord total cell protein lysates from the same subjects were also found to have a reduction in the fragmentation of the DNA repair protein poly(adenosine diphosphate-ribose) polymerase, indicating a reduction in neuronal apoptosis. This finding was coupled with histologic evidence of decreased neuronal damage and reduced neutrophil infiltration into the injured spinal cord at the zone of ischemic injury 48 hours after spinal cord ischemia [4].
Based on the efficacy of ATL-146e in preventing paralysis after spinal cord I/R, we inferred that the mechanism of neuronal salvage was founded on a decrease in reperfusion inflammation—an effect attributable to ATL-146e in treatment of reperfusion injury in other organ systems [5, 6]. In the current study we examined tissue samples from animals that underwent spinal cord I/R, and sought to reveal alternative mechanisms of action, whereby A2A-R activation on spinal cord neurons may serve a direct protective effect during the physiologic stress of I/R. This concept is supported by the observations of Kobayashi and Millhorn [7], who found that A2A-R expression could be stimulated by hypoxia in cultured pheochromocytoma cells. They found that A2A-R induction may enhance neuronal survival after physiologic stress, possibly by means of A2A-R-mediated regulation of intracellular calcium, which established cellular quiescence. The hypothesis of our current experiment is that spinal cord I/R results in the induction of neuronal A2A-R expression. Identification of a unique inducible A2A-R would provide evidence for a direct, potentially beneficial effect of adenosine A2A agonists in spinal cord protection after ischemic injury.
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Material and methods |
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Animal surgery
All animal protocols were reviewed and approved by the Animal Review Committee of the University of Virginia. Animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" as described by the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (Washington: National Academy Press, 1996). Fourteen New Zealand rabbits (weight, 3.0 to 3.5 kg) were selected and acclimatized for a minimum of 3 days within our vivarium. Each was anesthetized with an intramuscular injection of xylazine (10 mg) and ketamine (100 mg). An ear vein catheter was placed for administration of additional medications and intravenous fluids. The animals were intubated, placed supine on a heated operating table, and ventilated with a mixture of 98% oxygen and 2% halothane. Core body temperature was carefully maintained at 36°C in all animals using a heating blanket, and by closing the abdominal cavity in a timely fashion during the treatment interval. An ear arterial catheter was placed into a marginal artery for continuous monitoring of arterial pressure. Heparin sodium (2,000 U) was administered intravenously and allowed to circulate for 5 minutes. During this interval, the abdomen was sterilely prepared and draped. A midline laparotomy was made and the viscera reflected to the right. After opening the retroperitoneum, the abdominal aorta and inferior vena cava were identified. These great vessels were collectively clamped with Satinsky clamps just distal to the left renal artery, and again proximal to the aortoiliac bifurcation. Each animal underwent 45 minutes of warm spinal cord ischemia, with the clamps removed just before closure. Pulse oximetry assured adequate oxygenation throughout the procedure. This technique exploits the highly segmental arterial supply to the lumbar spinal cord in rabbits, and has resulted in reproducible dense paralysis in prior studies in our laboratory.
Drug administration
Eight microliters of stock ATL-146e solution (5 µg/µL in dimethyl sulfoxide) was diluted in 4 mL of normal saline carrier. Using a Harvard pump, the compound (40 µg) was infused into an ear vein at a constant rate for 3 hours, beginning after 30 minutes of spinal cord ischemia. Control ischemic animals were given carrier (4 mL of normal saline) without drug. One group of animals (I/R + ATL, n = 8) received 0.06 µg · kg-1 · min-1 intravenous ATL-146e, infused more than 3 hours, beginning after 30 minutes of ischemic time. A second cohort (I/R, n = 6) received vehicle, and served as a control group. A third group (Sham, n = 4) underwent laparotomy under general anesthesia alone, and served as sham-operated animals. The timing of drug administration is based on the short plasma half-life of ATL-146e in rodents, seeking to establish a steady-state concentration by the early reperfusion interval. The optimal concentration of drug has been determined by dose-response curves generated in earlier experiments [4].
Physiologic testing
Postoperatively, animals were supported with food and fresh water. At 48 hours after operation, hind limb neurologic function was evaluated using the following modified Tarlov scoring system [8]: 0, atony; 1, slight movement; 2, sits with assistance; 3, sits alone; 4, weak hop; 5, normal gait or hopping. The same technician in a blinded fashion performed all neurologic functional evaluations.
Adenosine A2A receptor staining
At necropsy, lumbar sections of spinal cord from the zone of injury and corresponding cervical sections (remote from the ischemic zone) were fixed in 10% formalin and embedded in paraffin for sectioning. Two-micrometer-thick sections were affixed to glass slides, deparaffinized in xylene, and rehydrated in serial dilutions of ethanol. All specimen slides were processed simultaneously in the same rack with identical treatment techniques. Antigen retrieval was conducted using a microwave-heated unmasking solution (Vector Laboratories Inc., Burlingame, CA). After blocking, the sample sections were incubated with a 1:500 dilution of polyclonal goat anti-human A2A-R antibody (courtesy of Diane Rosin Okusa, PhD) overnight at 4°C. After washing in phosphate-buffered saline, sections were incubated with biotinylated horse anti-goat antibody (Vector Laboratories Inc.) for 1 hour at room temperature. Immunostaining was accomplished using a horseradish peroxidase system (Vector Laboratories Inc.) and diaminobenzidine chromogen (DAKO Corporation, CA). Counterstaining was performed using filtered hematoxylin. All sections were reviewed in a blinded fashion by the same trained observer and evaluated according to a scale of relative staining intensity (0 to 4). The scoring system was based on the number of neurons within a high-powered field that stained positively, and the relative staining intensity of each neuron. This observer-based technique was chosen on the basis of the negative staining of animals that did sustain spinal cord ischemia. If constitutive staining had been observed in normal spinal cord, the experimental samples could have been described as a percentage of control.
Statistical analysis
All results are expressed as the mean ± standard error of the mean. Data were analyzed for between-group differences using Student’s t test. Significance was defined as a p value less than 0.05, as determined using SPSS Software (SPSS Inc, Chicago, IL).
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Results |
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Adenosine A2A receptor staining
There was intense staining of neuronal cell bodies and surrounding axons in transverse sections of spinal cord from I/R rabbits (Figs 1 and 2).
Rabbits in the I/R + ATL group had reduced and variable expression of A2A-R, with subtle overall staining of neurons. Within both I/R + ATL and I/R groups, the distribution of stained neurons was most pronounced within the ventral horn of the spinal cord from the lumbar zone of ischemic injury. Sham-operated animals were remarkably devoid of A2A-R staining, and were indiscernible from negative control slides that received only biotinylated secondary antibody and horseradish peroxidase. Sections of cervical spinal cord (remote from the zone of ischemic injury) from I/R rabbits demonstrated reactivity similar in intensity to neurons seen within the zone of ischemic injury.
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Comment |
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Adenosine is normally present in the central nervous system, and can have inhibitory effects on neuronal activity that appear to be mediated by both adenosine A1 and A2A receptors [9]. This is further supported by the observation that caffeine, an adenosine receptor antagonist, relieves tonic inhibitory activity. It is thought that adenosine-mediated neuronal quiescence may serve a neuroprotective effect, in addition to facilitating other central nervous system physiologic responses such as regulation of sleep, arousal, seizure activity, and the effects of ethanol and chronic drug use.
Adenosine is an endogenous purine that is abundant in normal tissue environments, and increases when released at times of hypoglycemia, hypoxemia, or traumatic tissue injury. Additionally there appears to be a stress response by injured cells to upregulate surface A2A-R expression in an inducible fashion. From the current experiment, we infer that the upregulation occurs within 48 hours of spinal cord ischemia. This cellular induction may constitute a preconditioning response that can be exploited by adenosine A2A agonists to salvage neurons and improve patient outcome after a tissue-threatening ischemic event. We are able to exploit this unique salvage mechanism through systemic administration of the potent adenosine A2A agonist ATL-146e. Interestingly, we found that staining for A2A-R is particularly pronounced in the ventral motor column, and correlates with preservation of hind limb motor function 48 hours after spinal cord ischemia.
The observation of A2A-R staining within cervical spinal cord after lumbar spinal cord I/R supports multiple possible hypotheses for A2A-R induction. Intrathecal hypoxia, oxygen-derived free radicals, or the presence of cytokines, such as interleukin 1 or tumor necrosis factor-
in the systemic circulation or in the cerebrospinal fluid could result in alterations in A2A-R expression by neurons throughout the central nervous system. Alternatively, intercellular interaction between lower and upper motor neurons may induce a generalized response by the central nervous system to express A2A-R after I/R. The current study suggests that treatment of rabbit subjects with ATL-146e during reperfusion results in a relative reduction of A2A-R expression by neurons within the ventral horn of the spinal cord. This effect was observed in sections from the lumbar zone of ischemic injury and within cervical spinal cord regions. This observation is supported by the findings of Kobayashi and Millhorn [7] that hypoxic pheochromocytoma cells treated with adenosine in vitro demonstrate decreased A2A-R gene expression. Treated neurons are thought to decrease surface expression of A2A-R through negative feedback of the A2A-R gene. The absence of receptors therefore supports the efficacy of intravenous administration of ATL-146e, a highly potent and specific agonist for A2A-R. It cannot be confirmed from the current observations that A2A-R reduction is a result of gene regulation. An alternative explanation could be that ATL-146e occupies surface receptors for the entire reperfusion interval, and simply masks their positive staining by immunohistochemistry. The drug-receptor interaction remains a focus for further investigation.
The novel finding of the induction of A2A-R on neurons after ischemic injury supports a separate paradigm for neuroprotection that would include ischemic preconditioning with subsequent A2A-R activation, coupled through G proteins to increased adenylate cyclase activity, regulating intracellular calcium availability with attenuation of neuronal excitability [9, 10]. Such A2A-R stimulation may lead to neuronal quiescence by producing electrical silence during periods of physiologic stress. Therein may exist a pharmacologic rationale for using adenosine A2A-R agonists such as ATL-146e to enhance an endogenous mechanism for neuronal survival, and reduce spinal cord injury after thoracic aortic reconstruction [7].
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Supported by National Institutes of Health research grant R01 NS39499-01A1. Additional support by NICHD/NIH through cooperative agreement U54 HD28934 as part of the Specialized Cooperative Centers Program in Reproductive Research.
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Discussion |
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Doctor Cassada and his associates are to be congratulated for pointing out in this paper and some earlier publications the importance of reperfusion inflammation in spinal cord ischemic injury. I would wager that if you looked at the literature 4 or 5 years ago, you would not find this term reperfusion inflammation; your group is to be congratulated on emphasizing that particular area of damage.
The thing that impresses me very much about your study is the fact that you are able to start your agent, your adenosine A2A agonist, after 30 minutes of ischemia. Certainly there are a lot of studies out there that show with 20 minutes of cross-clamping in the rabbit, you can get severe spinal cord damage with poor Tarlov scores. So the fact that you could start your agent after 30 minutes is, I think, quite significant and points out the importance of the reperfusion inflammation and the fact that maybe we can go longer with cross-clamping if we have better methods of ameliorating the reperfusion inflammation.
And also of significance is the fact that you showed not only upregulation of the adenosine A2A receptors in the lumbar area of ischemia but also in the cervical area. Maybe you can just elaborate briefly on that.
And finally, with regard to your protective agent, ATL-146e, is this material going to be inert enough to be approved for clinical use, and if so, when would you expect the availability of this agent?
DR CASSADA: Thank you, Dr Gott, for reviewing our paper and offering these insightful comments and questions. The concept of reperfusion inflammation applies to a series of events occurring after reestablishing blood flow. We believe that these events are probably related to exposure of neuronal and glial elements to different soluble factors like the development of oxygen-derived free radicals or cytokines with subsequent endothelial-leukocyte activation. We have exploited this concept by using an adenosine A2A-specific agonist during the early reperfusion interval. This response can be graded if given from 1 to 3 hours after removal of the cross-clamp, indicating a time dependency of its effects.
Additionally we have shown that serum concentrations of tumor necrosis factor- also reach peak concentrations at roughly 1.5 to 2 hours after cross-clamp removal, and administration of this drug seems to ameliorate that cytokine. Additionally, at 48 hours, there is evidence of improved neuronal viability and a decrease in the active process of neuronal apoptosis, which may be a clinically relevant mechanism that explains why we see patients doing quite well 1 to 2 days after a thoracoabdominal aneurysm repair and then going on to develop neurologic deficits.
The observation of neurons in the cervical spinal cord upregulating adenosine A2A receptor expression after lumbar ischemia is a puzzling one. We think this may represent a ubiquitous central nervous system survival mechanism in response to ischemic injury stimulus, and there are several possible ways we think this could be explained. The first potential mechanism could be in response to the inflammatory cytokines, tumor necrosis factor or interleukin 1, or, alternatively, oxygen-derived free radical accumulation intrathecally within the cerebrospinal fluid. Other possibilities include lower to upper middle neuron communication, causing upregulation of proximal receptor expression. Perhaps intravascular cytokine levels may have something to do with this.
The drug is really a concept under development at the University of Virginia, and it is one of several analogs. Pharmaceutical companies have approached the university about acquiring it and developing it. The first US Food and Drug Administration trial of such drugs will possibly be under a label applied to treatment of reactive airways disease and inflammation, and there is conceivably some off-label application for paralysis prevention. ATL-146e is a powerful drug with a reasonably narrow therapeutic window and significant side effects, and some other analog may come along before this becomes clinically available that has a more tolerable side effect profile. An analog with a longer half-life could be given as a bolus rather than a drip, and that may be the manifestation of this research that we see come to a clinical fruition.
DR ANTHONY L. ESTRERA (Houston, TX): Again, Dr Cassada, I would like to congratulate you on a very nice study. This is very important work in trying to understand the pathophysiology of neurologic deficits and paraplegia. In reviewing Dr Safi’s experience during the past several years with the use of the adjuncts distal aortic perfusion and cerebrospinal fluid drainage, we have seen a decrease in immediate paraplegia, but at the same time we have also seen a relative increase in delayed paraplegia. Others have also written about this concept of delayed paraplegia. Doctor Sundt’s group will be presenting a paper in the upcoming Society of Thoracic Surgeons meeting. I would like your thoughts about the complication of delayed paraplegia and the benefits of this type of treatment for delayed paraplegia.
Again, I think this is an important work in the understanding of the pathophysiology of neurologic injury after thoracic aortic operation, and might give us insight about postischemic inflammation as a pathologic mechanism for delayed paraplegia.
Thank you.
DR CASSADA: Adenosine has been evaluated in multiple organ systems looking at reperfusion injury, and the uniform observations made in different laboratories conclude that it decreases adhesion molecule expression on the endothelium, decreases leukocyte diapedesis to extravascular sites of injury, and in some cases decreases tumor necrosis factor- elaboration. All of these inflammatory events are apparently most intense early during the reperfusion process.
There seems to be some early initiating event that sets cells on a course of programmed cell death. There have been some authors who write about a tumor necrosis factor unique receptor on neurons that can be activated and will initiate these events. So tumor necrosis factor seems to be a very important early factor, and we know from prior studies that it is elevated immediately after the reperfusion begins and may return to basal levels quickly. Our goal is to somehow intervene during that critical period with drugs that may blunt that effect, preventing delayed effects related to attrition of neurons secondary to the active process of apoptosis.
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