Ann Thorac Surg 2000;69:792-798
© 2000 The Society of Thoracic Surgeons
Original Articles
Lazaroid reduces production of IL-8 and IL-1 receptor antagonist in ischemic spinal cord injury
Takashi Kunihara, MDa,
Shigeyuki Sasaki, MDa,
Norihiko Shiiya, MDa,
Hiroshi Ishikura, MDb,
Yo Kawarada, MDb,
Akihiro Matsukawa, MDa,b,
Keishu Yasuda, MDa
a Department of Cardiovascular Surgery, Hokkaido University School of Medicine, Sapporo, Japan
b First Department of Pathology, Hokkaido University School of Medicine, Sapporo, Japan
Address reprint requests to Dr Sasaki, Department of Cardiovascular Surgery, Hokkaido University Hospital, N-14, W-5, Kita-ku, Sapporo, Japan 0608648
e-mail: sasakish{at}med.hokudai.ac.jp
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Abstract
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Background. 21-aminosteroids (lazaroids) have demonstrated the protective effect against cerebral ischemic injury through the inhibition of lipid peroxidation. We examined whether lazaroids affected the production of proinflammatory and antiinflammatory cytokines in ischemic spinal cord injury model.
Materials. Anesthetized New Zealand white rabbits underwent a 20-minute infrarenal aortic cross-clamping (AXC) with pretreatment of either an intravenous 3 mg/kg lazaroid U74389G (group L; n = 10) or the same volume saline (group P; n = 10). Sham operation group (group S; n = 6) underwent only exposure of the aorta. Plasma concentrations of interleukin (IL)-8, -1ß, -1 receptor antagonist (IL-1ra) and tumor necrosis factor (TNF)-
were measured at four time points. Functional assessment with Tarlov score at 24 and 48 hours after pretreatment, pathologic assessment of the spinal cord, and measurements of cytokine levels in the spinal cord were performed.
Results. The maximum elevation of plasma IL-8 and -1ra levels occurred at 1 hour after declamping in four measurement points. Plasma IL-8 and -1ra levels in group L were significantly lower than those in group P (*p < 0.05). Plasma TNF peaked at 5 minutes after declamping, but decreased afterwards. Plasma TNF levels were not different among three groups. Spinal IL-8 levels in group L (0.98 ± 0.34 ng/g tissue) were lower than those in group P (7.26 ± 2.26 ng/g tissue)(*p < 0.05). Spinal IL-1ra and TNF were not significantly different. Tarlov score and pathologic assessment were better in group L.
Conclusions. Lazaroid U-74389G reduced the production of systemic IL-8 and -1ra and spinal IL-8 when AXC caused spinal cord injury. These results indicate that lazaroids may attenuate ischemic endothelial cell injury or activation of leukocytes.
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Introduction
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Spinal cord injury after surgical repair of the descending thoracic or thoracoabdominal aortic disease remains a persistent clinical problem. The reported incidence of neurologic injury for the most high-risk Crawford group II aneurysms ranges from 30% to 40% [1–2]. In spite of the numerous clinical and laboratory efforts to avoid the devastating consequence, paraplegia continues to be a major postoperative complication. Mechanisms of spinal cord injury consist of inadequate blood supply and ischemia-reperfusion injury of the spinal cord [3]. The inadequate blood supply is most likely due to either inadequate collateral blood flow during aortic cross-clamping or failure to reconstruct segmental arteries that are critical to the spinal cord. Spinal cord ischemia in the perioperative period can result from perioperative hypotension, distal aortic hypotension after aortic cross-clamping, the interruption of critical intercostal and lumbar arteries, and thrombosis and embolism of intercostal arteries [3]. Moreover, reperfusion with oxygenated blood after ischemia would aggravate the spinal cord injury. In general, the process of ischemia-reperfusion injury consists of hypoxic endothelial cell activation, leukocyte-endothelial cell interactions, and neutrophil-mediated injury [4]. The endothelium has been shown to play a key role in the injury suffered after ischemia and reperfusion [4].
Previous studies on ischemia-reperfusion injury in the central nervous system have demonstrated a remarkable protective effect of lazaroids, a new series of 21-aminosteroids. These compounds have a potent inhibitory activity of iron-dependent lipid peroxidation [5–6] and have been shown to be beneficial in various settings of ischemia-reperfusion injury [7–9]. In the pathogenesis of ischemia-reperfusion injury the endothelium has been shown to play an important role [4], but little is known about whether lazaroids reduce the postischemic endothelial cell injury. The purpose of this study is to determine whether lazaroid U-74389G affects the production of proinflammatory and antiinflammatory cytokines in the rabbit spinal ischemic injury model, which may serve as a marker of the endothelial cell injury under ischemic conditions.
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Material and methods
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New Zealand white rabbits weighing 2.5 to 3.7 (mean 3.2 ± 0.1) kg were used in this study, and obtained from Hokudoh Animal, Inc (Hokkaido, Japan). All experiments are approved by the Hokkaido University School of Medicine Animal Care and Use Committee, and conform to the U.S. National Institutes of Health guidelines regulating the care and use of laboratory animals (National Institutes of Health publication No.85 to 23, revised in 1985). All experiments were performed in the Research Institute of Hokkaido University School of Medicine. Lazaroid U74389G (Upjohn, Kalamazoo, MI), a promising clinical candidate among lazaroids, was prepared in the stock solution by CS-4 formulation (20 mmol/L citric acid monohydrate, 3.2 mmol/L sodium citrate dihydrate, 77 mmol/L NaCl) with a final concentration of 2 mg/ml. Unless otherwise stated, all reagents were purchased from Takeyama, Inc (Hokkaido, Japan).
Experimental protocol
All New Zealand male white rabbits (n = 26) were premedicated with subcutaneous atropine sulfate (0.005 mg/kg) and ketamine hydrochloride (40 mg/kg) and anesthetized by an intravenous injection of sodium pentobarbital (2 mg/kg). Heparin (100 IU/kg) was administered intravenously into the ear vein. An endotracheal tube was introduced through a tracheostomy and animals were ventilated with 38° C humidified oxygen (0.5 to 1L/min) at a respiratory rate of 50 breaths per minute. The tidal volume was set at 10 mL/kg body weight with a positive end-expiratory pressure of 2.5 cmH2O (Harvard Rodent Ventilator, Harvard Apparatus, Natick, MA). Adequacy of ventilation was confirmed by blood gas analysis at 37° C using a Radiometer ABL-625 system (Copenhagen, Denmark). Complete paralysis were obtained by an intravenous injection of suxamethonium chloride (2 mg/kg) and maintained by an intermittent intravenous injection of pancuronium bromide (0.05 mg/kg). Lactate Ringer’s solution (10 mL · kg-1 · h-1) was continuously infused during the experiment.
After sterile surgical preparation, all rabbits underwent an infrarenal aortic cross-clamping (AXC) through left retroperitoneal approach for 20 minutes to produce spinal cord ischemia. All rabbits were pretreated with either an intravenous 3 mg/kg Lazaroid U74389G (group L; n = 10) or the same volume saline (group P; n = 10) before aortic cross-clamping (AXC). Rabbits in sham operation group underwent only exposure of the aorta and received the same volume saline without aortic cross-clamping (group S; n = 6). Heart rate from the electrocardiogram were continuously monitored and recorded throughout the operation. Mean arterial pressures proximal and distal to the abdominal aorta were monitored with the method described by Agee and associates [10]. Briefly, a long 20G argyle catheter (Unitika Inc, Amagasaki, Japan) was advanced through one femoral artery to a location 1 to 1.5 cm just above the left renal artery for measurements of mean arterial pressures proximal to the abdominal aorta. An additional 24G catheter was placed into the opposite femoral artery for measurements of those distal to the abdominal aorta. All rabbits were warmed with a body temperature controlling system for rodent animals (Asahi Plate Warmer TK-43, Asahi Denshi Inc, Osaka, Japan) to keep the rectal temperature more than 38° C throughout the operation time.
For the assessment of systemic endothelial cell injury, plasma concentrations of interleukin-1ß (IL-1ß), interleukin-8 (IL-8), interleukin-1 receptor antagonist (IL-1ra), and tumor necrosis factor alpha (TNF) were measured. IL-1ß, IL-8, and TNF were measured as proinflammatory cytokines and IL-1ra was measured as a representative antiinflammatory cytokine. Whole blood samples were taken at the following times in group L and P: T0 = immediately before administration of lazaroid or saline, T1 = 5 minutes before declamping of the aorta, T2 = 5 minutes after declamping of the aorta, and T3 = 1 hour after declamping of the aorta. Blood samples were immediately centrifuged in an Eppendorf centrifuge (Eppendorf-Netheler-Hiniz-GMBH, Hamburg, Germany) and plasma was separated for immediate freezing at -80° C for the later measurements. Samples from rabbits in sham operation group were taken at the same time course (T0 = immediately before administration of saline, T1 = 15 minutes after T0, T2 = 25 minutes after T0, and T3 = 1 hour and 20 minutes after T0) without AXC and stored in the same manner. Concentrations of IL-1ß, IL-8, and IL-1 ra were measured by specific enzyme-linked immunosorbent assays (ELISA), as previously described [11–13]. The lower detection limit was 30 pg/mL for IL-1ß and 100 pg/mL for IL-8 and IL-1ra. TNF activity was determined using a L929 cytotoxic assay, as described by Flick and Gifford [14]. Equivalent concentrations of TNF were determined by interpolation of the known concentrations of human TNF run simultaneously. The lower detection limit of this assay was 100 pg/mL.
At 24 and 48 hours after pretreatment with lazaroid or saline, the neurologic functions were evaluated according to Tarlov score as follows: 0 = paraplegia with no lower extremity motor function; 1 = poor lower extremity motor function (flicker of movement or weak antigravity movement only); 2 = some lower extremity motor function with good antigravity strength but inability to draw legs under body and/or hop; 3 = ability to draw legs under body and hop but not normally; and 4 = normal motor function. Immediately after the functional assessment, all rabbits were sacrificed and the spinal cord tissue was taken for the pathologic assessment and for the measurements of tissue IL-1ß, IL-8, IL-1ra, and TNF levels. Pathologic assessment of the spinal cord was performed with hematoxylin and eosin (H-E), Klüver-Barrera (K-B), and glial fibrillary acid protein (GFAP) staining. Tissue cytokine levels were measured by the same method as the plasma cytokines.
Statistical analysis
All values are presented as mean ± standard error of the mean (SEM). Statistical analysis for comparisons of continuous variables was performed using one-way analysis of variance (ANOVA) with post hoc pairwise comparisons (Tukey-Kramer). Statistical analysis for comparisons of IL-8, IL-1ra, IL-1ß, and TNF between the group was performed using repeated measures ANOVA. Kruskal-Wallis test and Mann-Whitney’s U test were used for nonparametric comparisons of the Tarlov score. A p value less than 0.05 was considered statistically significant.
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Results
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Plasma IL-8 levels increased to a maximum level at 1 hour after declamping (T3) during sampling points. Plasma IL-8 levels at T0 through T3 were 5.4 ± 5.0, 5.7 ± 1.5, 8.5 ± 2.0, 34.0 ± 10.3 (mean ± SEM, ng/mL) in group P, 0.9 ± 0.3, 1.6 ± 0.5, 1.6 ± 0.4, 6.6 ± 3.0 in group L, and 0.2 ± 0.2, 1.8 ± 0.7, 3.8 ± 1.3, 9.7 ± 2.3 in group S, respectively. Plasma IL-8 levels in group L were significantly lower than those in group P (*p < 0.02 by repeated measures ANOVA) and equivalent to those in group S (Fig 1). In comparison of overall plasma IL-8 levels according to the time course, those at T3 significantly elevated relative to those at T0 (*p < 0.02), T1 (*p < 0.02) and T2 (*p < 0.05; by paired t-test).
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Fig 1. Changes in plasma interleukin-8 (IL-8) levels at four sampling times. T0 = immediately before aortic cross-clamping (AXC), T1 = 5 minutes before declamping of the aorta, T2 = 5 minutes after declamping of the aorta, and T3 = 1 hour after declamping of the aorta. (For group S [sham operation group], T0 = at the time of exposure of the aorta, T1 = 15 minutes after T0, T2 = 25 minutes after T0, and T3 = 1 hour and 20 minutes after T0). (Group P > group L, group S [*p < 0.02 by repeated measures ANOVA]; T3 > T0, T1, T2 [ p < 0.05; by paired t-test]).
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Plasma IL-1ra levels also increased to a maximum level at 1 hour after declamping (T3) during sampling points. Plasma IL-1ra levels at T0 through T3 were 4.3 ± 3.5, 7.6 ± 3.5, 18.3 ± 5.2, 68.4 ± 16.1 (mean ± SEM, ng/mL) in group P, 4.8 ± 3.3, 6.4 ± 3.9, 9.5 ± 4.5, 33.1 ± 4.9 in group L, and 0.1 ± 0.1, 2.4 ± 1.5, 7.5 ± 3.0, 54.3 ± 7.9 in group S, respectively. Plasma IL-ra levels in group L were significantly lower than those in group P (*p < 0.05 by repeated measures ANOVA) (Fig 2). In comparison of overall plasma IL-ra levels according to the time course, those at T3 significantly elevated relative to those at T0 through T2 (***p < 0.0001; by paired t-test).
Plasma TNF peaked at 5 minutes after declamping (T2 > T3, *p < 0.02 by paired t-test), but decreased to pre-AXC levels afterwards (Fig 3). Plasma TNF levels were not different among three groups (not significant; by repeated measures ANOVA). Plasma IL-1ß was under the minimum detectable level in all groups.
Spinal IL-8 levels in group L (0.98 ± 0.34 ng/g tissue) were significantly lower than those in group P (7.26 ± 2.26 ng/g tissue, *p < 0.05 vs group L) and equivalent to those in group S (Fig 4). Spinal IL-1ra and TNF showed similar tendency but differences among the groups were not significantly different (p = 0.093 for IL-1ra and p = 0.566 for TNF) (Figs 5, 6 ).
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Fig 4. IL-8 levels in spinal cord tissue samples measured by the specific ELISA. (Group P > Group L, Group S [*p < 0.05]).
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The mean Tarlov scores at 24 hours were 0.0 ± 0.0 in group P, 3.6 ± 0.4 in group L, and 4.0 ± 0.0 in group S. Those at 48 hours were 0.0 ± 0.0 in group P, 3.0 ± 0.8 in group L, and 4.0 ± 0.0 in group S. Tarlov scores in group L were significantly better than those in group P (**p < 0.01 at 24 hours and *p < 0.02 at 48 hours by Mann-Whitney’s U test) and were equivalent to those in group S. There were no histopathologic abnormalities in sham-operated rabbits (group S). In saline-treated rabbits (group P), the majority of neurons shrank and showed a cytoplasmic condensation and an occasional loss of cytoplasmic Nissl substances. Some neurons in lazaroid-treated rabbits (group L) showed shrank changes, but the majority of neurons demonstrated well-preserved cytoplasmic Nissl substances and nuclear hematoxylin staining. Histopathologic findings in group L were better than those in group P (Fig 7).
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Fig 7. Hematoxylin and eosin staining (x 250). (A) Group S (sham operation); (B) group P (saline vehicle-treated rabbits); and (C) group L (lazaroid-treated rabbits).
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Changes with mean blood pressures during the measurement period are summarized in Table 1. During the AXC period, the mean blood pressures distal to the cross-clamp were significantly lower than those proximal to the cross-clamp in both group L and P. However, significant differences of blood pressure among the three groups were not seen during the measurement period.
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Comment
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In this study, lazaroids U74389G significantly reduced the elevation of plasma IL-8 and IL-1ra levels and spinal IL-8 levels when ischemic spinal cord injury occurred due to aortic cross-clamping. IL-8, a member of proinflammatory cytokines, is produced by a wide variety of cells in response to various type of stimulation [15]. Under ischemic conditions, IL-8 is released by endothelial cells and plays an important role for endothelial cell-neutrophil interactions [4]. IL-8 can increase the adhesiveness of endothelial cells for neutrophils [16] and is also important in the regulation of transendothelial neutrophil migration [4, 17]. Our results of measurements of IL-8 levels were summarized as follows: maximum increase in the plasma IL-8 levels was seen at T3, 1 hour after reperfusion in all groups; and pretreatment of lazaroids U74389G significantly reduced plasma and spinal IL-8 levels. Matsukawa and associates reported that IL-8 was not only a potent and selective neutrophil attractant, but is also a neutrophil activator in vivo [15]. Since the activated leukocytes can also produce IL-8 after reperfusion [4], the increase may be derived from either endothelial cells that suffered ischemia and reperfusion or activated leukocytes after reperfusion or both. Although ischemic spinal cord injury occurred with neurologic deficits in this model, the increase in the plasma IL-8 levels at T3 was not due to the spinal cord injury only. Since the infrarenal aortic cross-clamping and declamping cause the ischemia and reperfusion not only in the spinal cord, but also in the other organs, the elevation of plasma IL-8 levels may be derived from the ischemia and reperfusion in the other organs. To demonstrate the definite production of IL-8 in the spinal cord, tissue IL-8 levels in the postischemic spinal cord were measured in this study. Spinal IL-8 levels in the rabbits receiving vehicle were significantly elevated in comparison to those in sham operation group and those treated with lazaroids. These results indicated that IL-8 was produced in the postischemic spinal cord, in which either endothelial cell injury or activated leukocytes after reperfusion or both were present. Such locally elevated concentrations of IL-8 would be an essential signal showing the neutrophil-mediated injury pattern [4].
Lazaroids, a new series of 21-aminosteroids, have demonstrated a remarkable protective effect against cerebral ischemic injury through the inhibitory activity of iron-dependent lipid peroxidation [5–6]. These compounds have also shown beneficial effects of attenuating ischemia-reperfusion injury in a variety of experimental models [7–9]. It is speculated that lazaroids may also attenuate endothelial cell injury through the inhibition of lipid peroxidation, but little is known about whether lazaroids attenuate the postischemic endothelial cell injury or influence the production of cytokines. Recently, Salahudeen and associates showed that administration of lazaroid was associated with better graft function and reduced lipid peroxidation in the setting of renal transplantation. Compared with the normal control kidneys, the kidneys transplanted with vehicle had higher cytokine mRNA levels for IL-6 and TNF. The levels for these cytokines were reduced in kidneys transplanted with lazaroids [18]. Rabinovitch and associates have also shown that other type of lazaroid U78518E significantly decreased the cytokine-induced increase in islet malondialdehyde and protected islet beta cells from destruction by the cytokine combination of IL-1 and TNF [19]. Our new finding, that a reduction in the production of plasma and tissue IL-8 levels in the ischemic spinal cord injury model, may partly result from protective effects of lazaroid against endothelial injury.
Antiinflammatory cytokines such as IL-1ra or IL-10 have been shown to have the effect of counterbalancing or controlling the proinflammatory response [20]. IL-1 is a proinflammatory cytokine that exerts a wide range of biological effects, and the action of IL-1 is regulated by their receptor expression, downregulation and shedding, and by inhibitory cytokine IL-1ra [21–22]. IL-1ra is generally produced and released by endothelial cells and leukocytes, in response to the increase in IL-1, IL-8 [15], or surgical stress [20]. In our study, maximum increase in the plasma IL-1ra levels was seen at T3, 1 hour after reperfusion as well as plasma IL-8 levels. There may be two possible reasons that account for the increase in the IL-1ra levels. First, the increase in the plasma IL-8 levels might stimulate the induction of IL-1ra, as is reported by Matsukawa and associates [15]. In this case, the increase in the IL-8 levels is followed by the increase in the IL-1ra levels. Second, IL-1ra levels might elevate in response to surgical stress. Atwell and associates have found the similar pattern in measurements of plasma IL-1ra levels in patients undergoing thoracic operation [20]. Plasma IL-1ra levels in their study became significantly elevated according to the operation time course. Another finding in this study is that pretreatment of lazaroids U74389G significantly reduced plasma IL-1ra levels. This finding may also be explained by the fact that lazaroids U74389G significantly reduced the production of IL-8, which stimulates the induction of IL-1ra. IL-1ra may be released to counterbalance the effect of increase in the proinflammatory cytokines.
IL-1ß was not detected in this study. Since IL-1ß production can stimulate the production of IL-8, the increase in the IL-8 levels may be preceded by the increase in IL-1ß levels [15]. Atwell and associates have also found that detectable plasma IL-1ß was not present in their study [20]. As endogenous IL-1ra may regulate part of the IL-1 activity, the IL-1ß activity was possibly reduced in the presence of IL-1ra.
TNF is a proinflammatory cytokine, which is mainly released by macrophages during ischemia and reperfusion. Plasma TNF levels peaked at 5 minutes after declamping, but decreased pre-AXC levels at 1 hour after reperfusion in our study. Since TNF may serve as an initiator in the kinetics of the generation of cytokines [13] and may induce the production of IL-8 [15], the increase in plasma TNF levels might promote the increase in plasma IL-8 levels in our study. However, significant difference of plasma TNF levels was found only between those at T2 and T3, and no difference was noted among the 3 groups. Thus significant difference of IL-8 production cannot be attributed to the difference of TNF production in our study. Possibly, lazaroids have no inhibitory effects on the release of TNF from macrophages or activated leukocytes.
There is no new finding in the neurologic and pathologic assessment in this study. The evidence that lazaroids attenuate postischemic spinal cord injury has been shown previously [23–25]. Neurologic and pathologic findings in our study may validate the adequacy of our experimental model. The ischemic time of infrarenal aortic cross-clamping in this study was set at 20 minutes because the mean Tarlov scores at 24 hours were most significantly different between the treated and untreated groups among various ischemic periods according to preliminary studies. We designed this study to investigate the difference of proinflammatory and antiinflammatory cytokine production when clinical symptoms contrasted strikingly between the groups of treated and untreated animals. However, it would be beneficial to measure IL-8 and other cytokine levels in cord tissue at earlier intervals of ischemia and reperfusion for further elucidation of cytokine production in the postischemic spinal cord injury, not only in the irreversible paraplegia but also in the paresthesia, reversible neurologic deficits.
It is suggested that smooth muscle cell proliferation caused by proinflammatory cytokines may be involved with the occurrence of delayed spinal ischemic injury [26–27]. Proinflammatory cytokines may serve as a marker of endothelial injury under ischemic conditions, thus measurements of cytokines would be useful in the assessment of the severity of ischemic organ injury. In addition, reduction in these cytokine levels itself may be beneficial for the protection against delayed spinal ischemic injury. Further studies will be required to elucidate that mechanism.
In conclusion, administration of lazaroid U-74389G before aortic cross-clamping significantly reduced the production of systemic IL-8 and IL-1ra when aortic cross-clamping caused spinal cord injury. Production of IL-8 in the postischemic spinal cord tissue was also reduced. These results indicate that lazaroids may attenuate endothelial cell injury or activation of leukocytes in the spinal cord that suffered ischemia and reperfusion.
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Acknowledgments
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Supported in part by grant-in-aid #08457338 for scientific research, from the Japanese Ministry of Education.
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Accepted for publication August 28, 1999.
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Abstract
Introduction
