Resveratrol, a Natural Red Wine Polyphenol, Reduces Ischemia-Reperfusion Induced Spinal Cord Injury

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1 Resveratrol, a Natural Red Wine Polyphenol, Reduces Ischemia-Reperfusion Induced Spinal Cord Injury Sadi Kaplan, MD, Gianluigi Bisleri, MD, Jeffrey A. Morgan, MD, Faisal H. Cheema, MD, and Mehmet C. Oz, MD Department of Surgery, Division of Cardiothoracic Surgery, College of Physicians and Surgeons, Columbia University, New York, New York Background. Severe neurologic injury still represents one of the most devastating complications after surgical repair of thoracoabdominal aneurysms. We therefore aimed to investigate the protective effect of resveratrol, a natural polyphenol antioxidant present in grapes and wine, in an experimental model of spinal cord ischemiareperfusion injury. Methods. Sixteen rabbits were assigned either to group A(n 8; receiving resveratrol, treated group) or group B (n 8; control group, nontreated group) and underwent a 30-minutes period of spinal cord ischemia by clamping the abdominal aorta between the left renal artery and the aortic bifurcation. Fifteen minutes before clamping, rabbits received either intravenous resveratrol (100 g/kg; group A) or normal saline (group B). Functional assessment with Tarlov score at 8, 16, and 24 hours postoperatively, histopathologic assessment of the spinal cord, measurements of malondialdehyde levels, and myeloperoxidase activity in the spinal cord were performed. Results. Neurologic impairment (Tavlov score for group A and for group B , p < 0.001), malondialdehyde levels ( nmol/g versus nmol/g, p < 0.001), and myeloperoxidase activity ( nm/min versus nm/min, p 0.002) were significantly lower in the resveratrol-treated animals. Additionaly, pathologically assessed outcomes were better in the resveratrol-treated group. The total number of motor neurons in the gray matter was significantly lower in the nontreated group than in the resveratrol-treated group ( versus , p 0.003). Conclusions. Prophylactic use of resveratrol reduced neurologic injury and provided clinical improvement by attenuating the inflammatory milieu in the rabbit spinal cord ischemia/reperfusion model. (Ann Thorac Surg 2005;80:2242 9) 2005 by The Society of Thoracic Surgeons Accepted for publication May 9, Address correspondence to Dr Kaplan, 2 Dedeefendi Altay Sokak 4/11, Kurtulus, Ankara 06600, Turkey; address: skaplan@bir.net.tr. Despite recent advances in operative techniques, anesthetic management, and postoperative care, paraplegia still remains one of the most devastating complications after surgery of thoracoabdominal aneurysms, ranging in incidence from 2.9% to 32% [1]. Optimal protection of the spinal cord from ischemia-reperfusion (IR) injury is the cornerstone for success with thoracoabdominal aortic surgery. Therefore, different operative and nonoperative strategies have been developed to increase spinal cord tolerance to ischemia and minimize the incidence of neurologic complications after aortic surgery [2, 3]. Mechanisms of spinal cord injury are complex and multifactorial. In general, the process of IR injury consists of inadequate blood supply, hypoxic endothelial cell activation, leukocyte-endothelial cell interactions, and neutrophil-mediated injury [1, 4]. The endothelium has been shown to play a key role in the injury suffered after ischemia and reperfusion. Energy failure, excitotoxicity, and oxidative stress have been strongly implicated in the pathogenesis of neurologic injury after spinal cord ischemia [4, 5]. Neutrophil activation and oxidative stress lead to massive production of free O 2 radicals, which in turn enhances inflammatory processes, such as lipid peroxidation, protein damage, and DNA damage [5 7]. As a consequence, suppression of neutrophil activation and lipid peroxidation by decreasing oxidative stress appears to be an important mechanism in the protection of the spinal cord from IR injury [5, 6]. Resveratrol (3,5,4 -trihydroxy-stilbene), a natural polyphenol antioxidant present in grapes, and the active constituent of red wine, has been causatively associated with the cardiovascular benefits associated with moderate wine consumption (the so-called French paradox ) [8, 9]. In addition to its vasodilatory effects [9], resveratrol exerts significant antioxidant [9, 10], antiplatelet [10], and anti-inflammatory [11] effects. Moreover, resveratrol was recently found to enhance nitric oxide (NO) production in various organs, such as endothelial cells and heart [9, 12 14], and consequently to exert a beneficial effect on several organs in IR injury [9 14]. We therefore sought to investigate whether intravenous administration of resveratrol could have a protective role in an experimental model of spinal cord IR injury. To assess the ability of resveratrol to prevent 2005 by The Society of Thoracic Surgeons /05/$30.00 Published by Elsevier Inc doi: /j.athoracsur

2 Ann Thorac Surg KAPLAN ET AL 2005;80: RESVERATROL REDUCES IR-INDUCED INJURY 2243 Table 1. Operative Data: Heart Rate, Mean Proximal and Distal Aortic Pressure, Esophageal Temperature, Arterial Blood Gas, and Glucose Levels in Rabbits Variable Control Resveratrol Heart rate (beats/min) Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion Proximal MAP (mm Hg) Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion a Distal MAP (mm Hg) During aortic occlusion (10 minutes) a Esophageal temperature ( C) Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion ph Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion PO 2 (mm Hg) Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion PCO 2 (mm Hg) Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion Base excess Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion Glucose (mg/dl) Baseline (before drug treatment) Immediately before aortic occlusion During aortic occlusion (10 minutes) minutes after reperfusion hours after operation a p 0.05 versus control group. Data are presented as mean SD. MAP mean aortic pressure. neurologic injury, we evaluated the following outcome variables in rabbits: hind-limb motor function, histopathology (in particular the number of motor neurons in the spinal cords), and the effects of resveratrol against oxidative stress and neutrophil activation. The latter was assessed by measuring spinal cord tissue malondialdehyde levels and myeloperoxidase activity. Material and Methods Study Design Sixteen male New Zealand white rabbits (weight: kg) were used according to a protocol reviewed and approved by the Institutional Animal Care and Use Committee at Columbia University (protocol no:

3 2244 KAPLAN ET AL Ann Thorac Surg RESVERATROL REDUCES IR-INDUCED INJURY 2005;80: Fig 1. Mean neurologic scores of animals demonstrated a significant difference (p 0.001) between control and treatment groups at each time point (8 hours, 16 hours, and 24 hours). AAAA2127), in accordance with guidelines of the American Association for the Accreditation of Laboratory Animal Care. All experiments were performed in the Research Institute of Columbia University. In this experiment, we used a rabbit model for spinal cord ischemia, because of its unique segmental arterial blood supply to the spinal cord from the infrarenal aorta. Fig 3. Total number of motor neurons in the gray matter. Chemicals Resveratrol at 99% purity was purchased from Sigma Chemical (St. Louis, Missouri). Resveratrol solution was prepared freshly in a sterile manner with normal saline, avoiding exposure of the solution to light before its use. Surgical Procedure Preoperatively, the animals had access to tap water and food without restrictions. Rabbits were anesthetized by intramuscular injection of xylazine (5 mg/kg) and ketamine hydrochloride (35mg/kg), and maintained with a continuous inhalation of isoflurane (2%). After ensuring adequate depth of anesthesia, a 24G ear vein catheter was placed for administration of intravenous fluids and additional medications. An ear arterial catheter was placed to monitor arterial blood pressure and obtain blood samples for analysis. The animals were then intubated with an endotracheal tube and ventilated at a respiratory rate of 50 breaths per minute (Ohmeda VMC Fig 2. Hematoxylin and eosin staining (magnification, 100). (A) Group A, resveratrol-treated rabbits. (B) Group B, nontreated rabbits. Fig 4. Effect of resveratrol pretreatment on spinal cord neutrophil accumulation in rabbits 24 hours after surgery. Myeloperoxidase (MPO) activity ( Abs 460 nm/min) was determined to quantify neutrophil deposition in the ischemic reperfused spinal cord tissue.

4 Ann Thorac Surg KAPLAN ET AL 2005;80: RESVERATROL REDUCES IR-INDUCED INJURY 2245 Fig 5. Malondialdehyde (MDA) levels of lumbar spinal cord segments 24 hours after reperfusion. Anaesthesia Machine, Ohmeda, England) with a mixture of 100% oxygen and 2% isoflurane. An esophageal probe (Datascope, Paramus, New Jersey) and warming blanket were used to record and support core temperature, respectively. Finally, an additional 24G catheter was placed into the femoral artery for measurements of arterial blood pressure distal to the abdominal aorta. Cefazolin was injected intravenously with single dose at 10 mg/kg, and normal saline solution (20 ml kg 1 h 1 ) was continuously infused during the experiment. After sterile surgical preparation, the abdomen was opened with a midline laparotomy incision and the retroperitoneal abdominal aorta was exposed. A careful dissection of the abdominal aorta was carried out immediately caudal to the origin of the left renal artery and proximal to the aortic bifurcation. Heparin sodium (100 IU/kg) was administered intravenously before aortic cross-clamping. Heart rate was continuously monitored (Datascope PasportXG; Datascope, Paramus, New Jersey) throughout the operation. Similarly, mean arterial blood pressures proximal to the abdominal aorta (through the marginal ear artery) and mean arterial blood pressure distal to the abdominal aorta (through the femoral artery) were recorded. Finally, rabbits in group A (treated group) received an intravenous injection of resveratrol (100 g/kg) while group B (nontreated group) received the same volume of normal saline. We administered resveratrol 15 minutes before occluding the aorta so that drug could be evenly distributed throughout the circulation before we created ischemia. Fifteen minutes after resveratrol or saline administration, the abdominal aorta was cross-clamped using atraumatic vascular clamps immediately caudal to the left renal artery and above the aortic bifurcation. After 30 minutes of ischemia, the clamps were removed, allowing for reperfusion. The 30 minutes of ischemic insult is based on the results of our previous experiments with this model [15]. The abdomen was then closed. Of note, before laparotomy and before closure of the laparotomy, animals were given buprenorphine (0.01 mg/kg/im [Reckitt and Coleman, Inc, Delaware]) for complete pain relief. The animals were allowed to recover from anesthesia before being returned to the holding area, where they could move freely in their cages and were provided with food and water ad libitum. Arterial blood samples were taken at the following times in groups A and B: T0 baseline (before drug/ saline treatment), T1 immediately before aortic occlusion, T2 during aortic occlusion (10 minutes), T3 30 minutes after reperfusion, and T4 24 hours after reperfusion. Similarly, arterial blood gases and glucose levels were measured. Assessment of Neurologic Function The neurologic function of the animals was evaluated by one member of the research team, without knowledge of the treatment group, at 8, 16, and 24 hours after operation according to the Tarlov score as follows: 0 complete paralysis; 1 minimal movement; 2 standing with assistance; 3 standing alone; 4 weak hop; 5 normal hop. Histopathology and Analysis of Biochemical Markers of Neurologic Damage Immediately after the functional assessment, the rabbits were anesthetized by intramuscular injection of xylazine (5 mg/kg) and ketamine hydrochloride (35 mg/kg). The marginal ear vein and artery were cannulated and arterial blood samples were obtained for analysis. After achievement of adequate depth of anesthesia with sodium pentobarbital (50 mg/kg, administered intravenously), all rabbits were sacrificed and the spinal cord specimens were taken for pathologic assessment as well as for measurements of biochemical markers of neurologic damage. As a marker of oxidative stress and free O 2 radical mediated damage, malondialdehyde levels were measured at the lumbar segments of spinal cords removed from sacrificed animals 24 hours after reperfusion. Malondialdehyde levels were measured in spinal cord tissue according to the method we previously described [15]. Data were calculated in nmol/g protein. Accumulation of neutrophils in the ischemic tissue has been thought to be a major mechanism responsible for reperfusion injury. Therefore, myeloperoxidase activity, an enzyme occurring almost exclusively in neutrophils, was measured to determine the neutrophil recruitment to the spinal cord during the IR period. Myeloperoxidase activity was determined using the method of Mullane and associates [16]. Myeloperoxidase enzymatic activity was measured spectrophotometrically at 460 nm using a Power Wave-X microplate reader (Biotek Instruments, Winooski, Vermont). The results were expressed as Abs 460 nm/min. Malondialdehyde and myeloperoxidase values were expressed as the mean SD of duplicate determinations, and all assays were measured without prior knowledge as to the group of origin of each rabbit. Histopathologic assessment of the spinal cord was performed with hematoxylin and eosin staining. Serial transverse sections (5 m) were obtained at the L4-L5 level and stained with hematoxylin and eosin for evalu-

5 2246 KAPLAN ET AL Ann Thorac Surg RESVERATROL REDUCES IR-INDUCED INJURY 2005;80: ation. In 5 serial sections of each rabbit, the total number of motor neurons in the gray matter was counted and averaged. Statistical Analysis All statistics were performed using SPSS statistical software (release 10.0; SPSS, Chicago, Illinois). All values are presented as mean SD. Statistical analysis for comparisons of continuous variables in the groups was performed using Friedman two-way analysis of variance (ANOVA). Post hoc pairwise comparisons were made by Wilcoxon paired signed ranks test with Bonferroni correction. Comparisons between groups were made by Mann-Whitney U test. A p value less than 0.05 was considered statistically significant. Results Perioperative Data No adverse events occurred during surgery. Operative data are shown in Table 1. Heart rate, esophageal temperature, arterial blood gases, and glucose levels were similar in the two groups at each time point. After 30 minutes of reperfusion, proximal aortic blood pressure was significantly higher in the resveratrol-treated group (group A versus group B mm Hg, p 0.043). Furthermore, distal aortic blood pressure (residual arterial pressure) after 10 minutes of crossclamping was significantly higher in the reveratroltreated group (group A mm Hg versus group B mm Hg, p 0.008). Neurologic Functional Evaluation As depicted in Figure 1, the mean Tarlov scores at each time point were significantly higher in the resveratroltreated group that in the nontreated group, implying an improved functional neurologic outcome due to the use of resveratrol. At 8 hours, the Tarlov score was in the treated group and in the nontreated group, while at 16 and 24 hours, it was in the treated group and in the nontreated group. In the resveratrol-treated group, the neurologic status of all animals remained unaltered throughout the observational period as opposed to in the nontreated group, where 1 rabbit showed functional deterioration 8 hours after operation. In greater detail, 7 rabbits were in grade 5 and 1 rabbit in grade 3 at 8, 16, and 24 hours postoperatively in the resveratrol-treated group, as compared with in the nontreated group where 6 rabbits were in grade 0 and 2 rabbits in grade 2 at 8 hours, and 6 rabbits were in grade 0, 1 rabbit in grade 1, and 1 rabbit in grade 2 at 16 and 24 hours postoperatively. These result indicate that resveratrol protected the ischemic spinal cord from IR injury which translated into a clinical benefit. Histopathologic Evaluation Histopathologic findings in the treated group were better than those in the nontreated group (Fig 2). The total number of motor neurons in the gray matter was significantly lower in the nontreated group than in the resveratrol-treated group (group A versus group B , p 0.003; Fig 3). Additionally, in the nontreated group, there was evidence among the remaining motor neurons of shrinkage and chromatic agglutination, eosinophilic neuronal degeneration, inflammatory cell accumulation, swollen motor neuron cells, and vacuolization of gray matter. Conversely, in the resveratrol-treated group, large motor neurons with no evidence of histologic change and minimal inflammatory cell accumulation were observed in the anterior horn (Fig 2). Malondialdehyde Levels The malondialdehyde levels of resveratrol-treated groups were significantly lower than those of the nontreated group (group A nmol/g versus group B nmol/g; p 0.001; Fig 4), thus indicating decreased oxidative stress in presence of resveratrol. Myeloperoxidase Activity Spinal cord myeloperoxidase activity was significantly lower in the treated group as compared with the nontreated group (group A nm/min versus group B nm/min; p 0.002; Fig 5). This suggests that resveratrol may act as an inhibitor of neutrophil adherence to the endothelium of spinal cord during the IR period. Comment Surgery of the descending and thoracoabdominal aorta has been associated with post-operative paraparesis or paraplegia. The incidence of spinal cord ischemia and subsequent neurologic complications is primarily associated with the duration and severity of ischemia, as well as reperfusion injury [1, 5]. Therefore, different operative strategies have been developed in an attempt to minimize the incidence of neurologic complications after aortic surgery [1 3]. The endothelium has been shown to play a key role in spinal cord injury after IR. Additionally, oxidative stress has been significantly implicated in the pathogenesis of neurologic injury after spinal cord ischemia [4 7]. There is increasing evidence that free O 2 radicals are generated in substantial quantities by ischemia and reoxygenation of the energy-depleted cells, and that they contribute to tissue injury [7]. Unstable radicals are potent initiators of protein degradation and lipid peroxidation, which in turn can lead to cell membrane dysfunction and eventually cell death [5, 7]. These radicals also directly induce endothelial damage and cytokine production in brain cells via the activation of transcriptional factors, and coordinate with them to enhance neutrophil chemotactic mechanisms [6] Finally, free O 2 radicals produce lipid peroxidation as well as protein and DNA damage [5, 7] It has been demonstrated that free O 2 radical mediated lipid peroxidation is a selfperpetuating process that can spread to the circumferen-

6 Ann Thorac Surg KAPLAN ET AL 2005;80: RESVERATROL REDUCES IR-INDUCED INJURY 2247 tial undamaged neuronal tissue, leading to further collapse of microcirculation and to irreversible damage to myelin and axons [5]. We therefore investigated the potential protective properties of resveratrol, a polyphenolic compound present in grapes and red wine, that has recently been studied as an effective antioxidative agent [9, 10], especially with respect to its cardioprotective properties [8, 9]. Besides its antioxidant effect, several biological actions of resveratrol, such as its vasodilatory effects on blood vessels and anti-inflammatory effects, have been well documented [10, 11, 13]. Several studies in non neuronal organs, such as heart and kidney, have demonstrated the ability of resveratrol to reduce IR injury [17, 18]. In vivo antioxidant properties of resveratrol have been attributed to its stimulation of NO formation [10, 14] as well as its ability to act as a potent scavenger of free O 2 radicals generated by both neutrophils and the endothelium [11, 18]. Resveratrol-induced brain protection has been demonstrated by several authors. Wang and colleagues [19] demonstrated that resveratrol can cross the blood brain barrier and reduce the infarct size in rats subjected to focal cerebral ischemia. Huang and associates [20] showed that resveratrol, in lower doses than used in our experiment, can reduce infarct size in Long-Evans rats subjected to focal cerebral ischemia. Sinha and associates [21] demonstrated that chronic treatment with resveratrol for 21 days can significantly improve motor performance in rats as well as significantly decrease infarct volume. Wang and associates [22] demonstrated that resveratrol can decrease oxidative stress by increasing heme oxygenase activity and suppressing the inflammatory response through inhibition of interleukin-6 production in mixed glial cells. The neuroprotective effects of resveratrol has been attributed primarily to membrane protection by means of neutralization of free O 2 radicals [22], as well as through improvement of brain perfusion [20]. In our study, the spinal cord malondialdehyde levels (indicative of oxidative stress lipid peroxidation in spinal myelin) at 24 hours after aortic occlusion were significantly elevated in the nontreated group, implying the involvement of free O 2 radicals in neuronal injury. Conversely, we observed a less considerable increase in malondialdehyde content of spinal cord after IR when resveratrol was administred before aortic occlusion. Leukocyte-endothelial cell interaction and subsequent leukocyte adhesion to vascular endothelium, which is a crucial step in the cell-mediated damage, is important in the pathophysiology of spinal cord tissue injury. Expressed on endothelial cells, ICAM-1, VCAM-1, and P-selectin are important in leukocyte-endothelial cell interactions. They mediate the binding of leukocytes to endothelial cells through interactions with their counterreceptors on leukocytes [23]. Circulating leukocytes adhere to the vessel wall using these molecules and enter the tissue, release toxic substances, such as proteolytic enzymes and cytokines, generating free O 2 radicals and causing a considerable amount of damage to the vascular endothelium and adjacent tissues [23]. Furthermore, leukocyte recruitment to the ischemic tissue may contribute significantly to spinal cord injury by reducing microvascular blood flow, initiating postischemic microvascular thrombosis, and releasing free O 2 radicals [20, 24]. Ithas been demonstrated that endothelial free O 2 radical generation plays an important role in spinal cord injury by upregulating intercellular adhesion molecules and increasing leukocyte recruitment to the ischemic spinal tissue [24] Therefore, it is possible that attenuation of leukocyte adhesion to the vessel wall and infiltration to the ischemic tissue as well as decreasing oxidative stress during the IR period may be potentially beneficial in preventing spinal cord ischemia/repefusion injury. In our study, myeloperoxidase activity, as a marker of leukocyte activation in the ischemic tissue, increased after spinal IR in the nontreated group but was significantly reduced in the resveratrol-treated group. These data, which confirm the neuroprotective effects of resveratrol in spinal cord ischemia, suggest that the mechanism of this action may involve inhibition of leukocyte infiltration in the injured spinal cord. The results of the present study show that intravenous administration of resveratrol prior to ischemia had a protective effect on rabbit spinal cord motor neurons. Histopathology demonstrated that the total number of motor neurons in the gray matter was significantly lower in the nontreated group than that in the resveratrol-treated group. Additionally, in the nontreated group, there was evidence among the remaining motor neurons of shrinkage and chromatic agglutination, eosinophilic neuronal degeneration, inflammatory cell accumulation, swollen motor neuron cells, and vacuolization of gray matter. Finally, the histopathologic benefits of resveratrol treatment were confirmed during the neurologic functional assessment. The mean Tarlov scores at each time point were significantly higher in the resveratrol-treated group that in the nontreated group, implying an improved functional neurologic outcome due to the use of resveratrol. Spinal cord is a very sensitive organ to oxygen deprivation, therefore maintainance of blood flow to spinal cord is vital to maintain its integrity and functions. Interruption of blood flow to the spinal cord sets in motion molecular processes that prime the vasculature for amplification of the inflammatory response, which can rapidly lead to tissue injury on reestablishment of flow. Various components of the ischemic vascular milieu can potentially affect the subsequent reperfusion response including reduced oxygen tension, substrate deprivation, waste product accumulation, or ph. Various studies of the mechanisms underlying neuronal death due to spinal ischemia have indicated the importance of inadequate blood supply in this complex pathology. Eventually, exposure of spinal tissue to reduced blood supply initiates numerous biochemical events leading to loss of functional integrity and ultimately to cell death. Thus, factors or agents that modulate blood flow to ischemic spinal

7 2248 KAPLAN ET AL Ann Thorac Surg RESVERATROL REDUCES IR-INDUCED INJURY 2005;80: cord tissue during aortic crossclamping may be important in decreasing spinal cord injury formation after aortic crossclamping. Therefore, in this experiment, we measured proximal and distal aortic blood pressures during crossclamping and examined the impact of the vasodilatatory effects of resveratrol on the vasculature as well as it effect on spinal cord injury. It is interesting that in the resveratrol-treated group, whose spinal integrity was better preserved, there were higher residual arterial pressures during clamping compared with the nontreated group. It is possible that higher pressures during clamping promoted enhanced collateral flow to the ischemic segment of cord. This study is part of a set of experiments in which our group has focused on the protective effects of resveratrol on various tissues in different experimental models. Before conducting these experiments, we performed a detailed Medline research regarding resveratrol, and we examined the dosages of resveratrol used in previous experiments for brain protection and for the other organs. Before the current experiment, we investigated protective effects of resveratrol (at various concentrations) on heart and lung tissues in a mouse heart-lung transplantation model (presented at The European Society for Cardiovascular Surgery 2004 meeting, and papers are under evaluation), on the endothelium of arterial and venous coronary artery bypass grafts [25]. Thus, based on the encouraging results of our previous experiments with resveratrol, we selected this concentration in this experiment. The findings of this study indicate that resveratrol exerts protective effects on the spinal cord of rabbits at 100 g/kg. However, the dose of resveratrol used in this study may not be the optimal dose to obtain maximal resveratrol protection. Therefore, its optimal protective dose or dose range for spinal cord and for other tissues should be determined by conducting additional studies. In conclusion, preischemic infusion of resveratrol protects the spinal cord from IR injury in rabbits. This protection is probably related to decreased oxidative stress in the ischemic spinal cord tissue and decreased neutrophil infiltration. Moreover, the resveratrol-treated group had higher residual arterial pressures during clamping. This suggests that resveratrol treatment may have promoted enhanced collateral flow to the ischemic segment of spinal cord. Despite further studies being required for a more complete understanding of the underlying protective mechanism, correct dosage, and timing of administration, this natural antioxidant polyphenol may represent a useful tool in the armamentarium of surgeons for the prevention of neurologic deficits associated with surgical treatment of descending aortic and thoracoabdominal aortic pathology. This study was supported by Institutional Research Funds of Columbia University. Doctor Kaplan was supported by a research grant from the Turkish Ministry of Health. The authors wish to thank Erdem Karabulut, PhD, Department of Biostatistics, Hacettepe University, Ankara, Turkey, for the statistical analysis. References 1. Wada T, Yao H, Miyamoto T, et al. Prevention and detection of spinal cord injury during thoracic and thoracoabdominal aortic repairs. Ann Thorac Surg 2001;72: Coselli JS, LeMaire SA, Koksoy C, et al. Cerebrospinal fluid drainage reduces paraplegia after thoracoabdominal aortic aneurysm repair: results of a randomized clinical trial. J Vasc Surg 2002;35: Bisleri G, Tisi G, Negri A, et al. The bicircuit system: innovative perfusional options for surgical treatment of the thoracic aorta. Ann Thorac Surg 2005;79: Boyle EJ, Pohlman TH, Cornejo CJ, et al. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion. Ann Thorac Surg 1996;62: Ueno T, Furukawa K, Katayama Y, et al. Spinal cord protection: development of a paraplegia-preventive solution. Ann Thorac Surg 1994;58: Kirsch JR, Helfaer MA, Lange DG, et al. Evidence for free radical mechanisms of brain injury resulting from ischemia reperfusion induced events. J Neurotrauma 1992;9(Suppl 1): Agee JM, Flanagan T, Blackbourne LH, et al. Reducing postischemic paraplegia using conjugated superoxide dismutase. Ann Thorac Surg 1991;51: Constant J. Alcohol, ischemic heart disease and the French paradox. 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Pharmacological preconditioning with resveratrol: role of nitric oxide. Am J Physiol Heart Circ Physiol 2002;282:H Kaplan S, Ulus AT, Tutun U, et al. Effect of Mg2SO4 usage on spinal cord ischemia-reperfusion injury: electron microscopic and functional evaluation. Eur Surg Res 2004;36: Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Meth 1985;14: Giovannini L, Migliori M, Longoni BM, et al. Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. J Cardiovasc Pharmacol 2001;37: Ray PS, Maulik G, Cordis GA, et al. The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury. Free Rad Biol Med 1999;27: Wang Q, Xu J, Rottinghaus GE, et al. Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res 2002;958: Huang SS, Tsai MC, Chih CL, et al. Resveratrol reduction of infarct size in Long-Evans rats subjected to focal cerebral ischemia. Life Sci 2001;69: Sinha K, Chaudhary G, Gupta YK. Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats. Life Sci 2002;71: Wang MJ, Huang HM, Hsieh SJ, et al. Resveratrol inhibits interleukin-6 production in cortical mixed glial cells under

8 Ann Thorac Surg KAPLAN ET AL 2005;80: RESVERATROL REDUCES IR-INDUCED INJURY 2249 hypoxia/hypoglycemia followed by reoxygenation. Neuroimmunology 2001;112: Krieglstein CF, Granger DN. Adhesion molecules and their role in vascular disease. Am J Hypertens 2001;14: Suematsu M, Tamatani T, Delano FA, et al. Microvascular oxidative stress preceding leukocyte activation elicited by INVITED COMMENTARY Kaplan and colleagues [1] have found another pharmacologic intervention to protect the fragile spinal cord against ischemia reperfusion (IR) injury in a laboratory model. During the past 2 decades many drugs have been discovered to protect against such injury in animal studies, but time and again these agents have not been translated into clinical efficacy for humans. As much as we know about the pathophysiology of IR injury in numerous organ systems, an effective treatment for spinal cord IR injury eludes us. There is no doubt that the mechanisms discussed by the authors (ie, free oxygen radical formation and subsequent inflammation) play major roles in this particular IR injury. Resveratrol, a natural constituent of red wine, appears ideally suited to impact both of these major and interrelated phathophysiologic IR pathways as documented in this simple but elegant study. But will resveratrol ever make it to a clinical trial? Although the results of this study are very remarkable, there is much we do not know about resveratrol. What is the optimal dose in humans, let alone in this animal model? What is the optimal duration of treatment? Are the protective effects of resveratrol maintained beyond 24 hours? We all are aware of the toxicity of too much red wine. What is the toxicity profile of resveratrol? Studies such as this one by Kaplan and colleagues give us reason for hope, but we must temper that hope with the fact that many drugs have been here before. With respect to spinal cord IR injury, many drugs have shown promise in pre-clinical laboratory studies, only to never make it to clinical trials. Or if clinical trials have been done, the in vivo nitric oxide suppression. Am J Physiol 1994;266: H Kaplan S, Morgan JA, Bisleri G, et al. Effects of resveratrol in storage solution on adhesion molecule expression and nitric oxide synthesis in vein grafts. Ann Thorac Surg In press. results have been marginal at best. The fact is that no pharmacologic agent has been deemed the gold standard for significantly protecting the human spinal cord from IR injury. Although the rabbit is a well known and accepted model for spinal cord IR injury, perhaps the mechanism of human spinal cord IR injury is more complex. Impacting the oxidative stress and inflammation pathways may not be enough. Targeting other pathways of cell death (or repair and regeneration) may be equally or more important. Nevertheless, based on the amazing results of this investigation, resveratrol deserves more study. Should it prove effective, what a palatable new pre-medication selection we may have for our patients with thoracic aortic disease. John Kern, MD Department of Surgery University of Virginia Health Sciences Center Box Charlottesville, VA jak3r@virginia.edu Reference 1. Kaplan S, Bisleri G, Morgan JA, Cheema FH, Oz MC. Resveratrol, a natural red wine polyphenol, reduces ischemiareperfusion-induced spinal cord injury. Ann Thorac Surg 2005;80: by The Society of Thoracic Surgeons /05/$30.00 Published by Elsevier Inc doi: /j.athoracsur