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Anesth Analg 2003;97:857-862 © 2003 International Anesthesia Research Society
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Abstract |
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The pathophysiology of ischemia/reperfusion injury involves extravascular migration of leukocytes from the bloodstream to the site of injury. Leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) play an important role in the recruitment of leukocytes to the site of injury. In this study, we evaluated the role of the ICAM-1 in spinal cord ischemia and the therapeutic effects of epidural ICAM-1 monoclonal antibody (Mab). The descending aorta was occluded below the renal artery with an aneurysm clip in rabbits anesthetized with halothane. The following variables were evaluated, in addition to ICAM-1 expression in the lumbar spinal cord, in animals receiving saline or ICAM-1 Mab via the epidural route: (1) leukocyte recruitment in the lumen of capillary vessels of the lumbar spinal cord (L6-7) at 8 h after 30 min of aortic occlusion and (2) neurological evaluation at 20 h after aortic occlusion of 10, 15, 17.5, 20, or 25 min. Paraplegia was graded with the following scale: Grade 0, no deficit; Grade 1, partial deficit; and Grade 2, complete paraplegia. Spinal cord ischemia increased the expression of ICAM-1 in the endothelium of spinal cord capillaries and led to capillary leukocyte recruitment and extravascular migration into the lumbar spinal cord parenchyma, which was ablated with epidural ICAM-1 Mab. Epidural ICAM-1 Mab reduced neurological deficits and offered neuroprotection. These findings demonstrate the involvement of the ICAM-1 pathway in spinal cord ischemia and the neuroprotective effects of epidural ICAM-1 Mab. Strategies to ameliorate spinal cord ischemia may entail the administration of leukocyte antiadhesion molecules into the neuraxial space.
IMPLICATIONS: Spinal cord ischemia increased intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte recruitment. Epidural administration of ICAM-1 monoclonal antibody ablated leukocyte recruitment and reduced neurological deficits.
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Introduction |
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Paraplegia is a devastating complication that affects approximately 5% to 33% of patients who undergo thoracoabdominal aneurysm surgery (1–3). Cellular damage from ischemia/reperfusion injury (IRI) contributes to the pathophysiology of spinal cord ischemia after aortic surgery (4). IRI is exacerbated by the generation of oxyradicals (5,6). All classes of macromolecules, including proteins, lipids, and nucleic acids, are susceptible to attack from the oxyradicals, which, over time, results in impaired cellular function. Leukocytes, which are activated by cellular adhesion molecules, are involved in the production of oxyradicals (7,8). Moreover, further damage to reperfused tissue occurs secondary to leukocyte adhesion as leukocytes physically obstruct capillaries, infiltrate central nervous system (CNS) tissue, and cause neurotoxicity (9).
Intercellular adhesion molecules (ICAMs), which include ICAM-1 and ICAM-2, are key players in leukocyte-endothelial cell interactions, in which leukocytes migrate from the blood to the site of inflammation via leukocyte adhesion (10). Therapeutic agents that interfere with leukocyte adhesion and recruitment reduce tissue inflammation and injury in the brain and other organs, including the heart, lung, and kidney (11–13). The interaction of the ICAM-1 pathway in relation to cerebral tissue destruction secondary to IRI has been extensively investigated in both humans and animals (14–18). However, only a few experimental reports have characterized the role of ICAM-1 in spinal cord tissue damage (19,20). The systemic administration of ICAM-1 monoclonal antibody (Mab) has been shown to improve motor function after traumatic and ischemic spinal cord injury. In this study, we sought to evaluate the role of the ICAM-1 pathway after spinal cord ischemia and to determine the effects of epidurally administered ICAM-1 Mab on leukocyte recruitment and neurological function after aortic occlusion in rabbits.
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Methods |
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Approval for use of rabbits was obtained from the University of South Florida Institutional Animal Care and Use Committee. The rabbit model of ischemic paraplegia was developed on the basis of the detailed information provided by DeGirolami and Zivin (4). The segmental distribution of spinal cord blood supply, feeble collateral blood supply, anatomy, and cytoarchitecture specific to rabbit makes it an excellent and reproducible ischemic paraplegia model. New Zealand White male rabbits, weighing 2.75 to 3 kg, were anesthetized with halothane and placed on a warming blanket. A midline low abdominal incision was made, the intestines were reflected, and the descending aorta was exposed at the left renal artery. An aneurysm clip was placed on the aorta just below the origin of the left renal artery. After 30 min of aortic occlusion, the aneurysm clip was removed and the incision closed. Sham-operated control animals were treated similarly, except that the aorta was not occluded. For experimental purposes in this study, rabbits were randomized to various durations of aortic occlusion (10, 15, 17.5, 20, and 25 min). All rabbits that underwent 30 min of aortic occlusion developed paraplegia.
The ICAM-1 expression in the lumbar spinal cord was evaluated by immunohistochemistry. The details of the procedure are as follows: 8 h after IRI, rabbits were perfused with normal saline and heparin to remove blood from blood vessels. The lumbar spinal cord was quickly dissected and frozen in liquid nitrogen with tissue-freezing medium (Triangle Medical Sciences, Durham, NC) and stored in an -80°C freezer until further processed. ICAM-1 Mab was donated by Robert Rothlein, PhD (Boehringer Ingelheim, Richfield, CT). Sections of 5-µm thickness were cut on a cryostat, picked up on coated slides, allowed to air-dry, and fixed in acetone at 4°C for 10 min. The sections were washed in phosphate-buffered saline (PBS) and incubated sequentially in 10% goat serum, Mab for ICAM-1, 0.3% H2O2 in PBS, biotinylated horse anti-mouse immunoglobulin (Vector Laboratories, Burlingame, CA), and avidin-biotin-horseradish peroxidase complex (Vector). Washes in PBS were performed between incubation steps. Immunoperoxidase reaction product was visualized with 3-amino-9-ethyl carbazole (Aldrich Chemical Co., Milwaukee, WI) and fixed in formal-acetate. The sections were counterstained with hematoxylin. Reagents from a staining kit for detection of goat immunoglobulin (Vector) were used to identify Factor VIII-related antigen. The 
CD-31 was used as a positive control for endothelial cells. The negative controls included substitution of relevant Mabs in the same concentration as primary antibodies, PBS, and normal goat serum for primary antibodies. All the samples were processed in batches by using the same reagents and protocol. The experiments were repeated three times for reproducibility. Three animals per group were used (e.g., sham-operated control animals, saline control post-IRI animals, and ICAM-1 Mab-treated post-IRI animals).
As described by Malinovsky et al. (21), under halothane anesthesia, an epidural catheter was placed via a nontraumatic route into the lumbar epidural space. Briefly, an incision was made at the depression between the apex coccis sacri and the first vertebral coccygea, allowing a PE-10 tube to gently slide rostrally until the tip reached the L6-7 interspace. The catheter was tunneled and secured before skin incision closure. Catheter position was confirmed at autopsy. After placement of the epidural catheter, animals were allowed to recover. Epidural saline or ICAM-1 Mab was administered 1 h before aortic occlusion.
Under halothane anesthesia, rabbits underwent aortic occlusion for 30 min; then the aneurysm clip was removed and the incision closed. Sham-operated control animals were treated similarly, except that the aorta was not occluded. Eight hours after IRI, rabbits were perfused with normal saline and heparin to remove blood from blood vessels. The perfusion was continued with Trump’s fixative (Electron Microscopy Sciences, Fort Washington, PA), which consisted of glutaraldehyde, formalin, and sodium cacodylate. Immediately after primary fixation with Trump’s fixative, the lumbar cord was dissected and stored in Trump’s fixative at 4°C overnight. Tissue samples were postfixed in 1% osmium tetroxide, dehydrated in graded series of acetone, and embedded in Epon-Araldite. The lumbar spinal cord segments were identified on thick sections and processed for electron microscopy by using the protocol described by Sutton et al. (22) Thick sections (0.3 µm) were cut and stained with toluidine blue. Thick sections were used for localization of the capillary vessels, and ultrathin sections were obtained for finer resolution. These sections were stained with uranyl acetate and lead nitrate. The grids were viewed on a Hitachi 7000 electron microscope in transmission mode. The experiments were repeated three times for reproducibility. Three animals per group were used (e.g., sham-operated control animals, saline control post-IRI animals, and ICAM-1 Mab-treated post-IRI animals).
Epidural saline or ICAM-1 Mab was administered 1 h before aortic occlusion. Rabbits were randomized according to treatment with epidural saline or ICAM-1 Mab at various ischemic durations of aortic occlusion (10, 15, 17.5, 20, and 25 min). Twenty hours after aortic occlusion, a blinded observer assessed the severity of paraplegia by using a paraplegia scoring scale (Table 1), which is a modification of the neurological grading evaluation used by Clark et al. (20).
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The probability of Grade 2 paraplegia (Prob [paraplegia]) was modeled as a logistic function of the independent variables time (duration of aortic occlusion) and ICAM-1 treatment (ICAM-1 group, 1; saline control, 0). That is, we assumed a model of the form
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The data allow for the estimation of ß values and the standard errors of those estimates. This, in turn, enabled us to assess the statistical significance of each independent variable and to construct graphs of Prob [paraplegia] as a function of time for each group. By fixing ICAM-1 treatment at 0 and then 1, setting Prob [paraplegia] equal to 0.50, and solving for time, we were able to estimate ET50, the effective time at which 50% of the animals had Grade 2 paraplegia for both groups. All statistical analyses were performed with SAS version 8.2 (SAS Institute, Cary, NC).
This analysis entails iterative fitting of a logistic function to construct a sigmoidal quantal dose-response curve representing a range of duration of aortic occlusion with respect to neurological function. Its utility as a method to determine therapeutic efficacy of a pharmacological drug in experimental models of CNS ischemia has been demonstrated (20,23). The ET50, which indicates the estimated length of ischemia time to produce Grade 2 paraplegia in 50% of the animals, was calculated. A pharmacological drug that shifts the curve to the right indicates therapeutic efficacy, signifying that animals treated with this drug, on average, will tolerate a longer ischemic duration.
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Results |
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The acute-phase response in animals 8 h after IRI, after 30 min of aortic occlusion, leads to an increase in ICAM-1 expression in the capillary endothelial cells in the lumbar cord (Fig. 1, A and B) compared with sham-operated controls. To verify ICAM-1 expression on endothelial cells, frozen-section samples obtained from rabbits 8 h after IRI were cross-reacted with
CD-31, a positive control for endothelial cells in the capillary blood vessels (Fig. 1C). Likewise, frozen-section samples were cross-reacted with negative control serum (Fig. 1D).
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The area of spinal cord that was used for comparison in this study was L6-7, which corresponded to the site of the epidural catheter tip. On the basis of previous work studying the caudal lumbar cord, this portion of the spinal cord has been shown to have localization of ischemic injury accompanied by increases in apoptosis and antioxidant enzyme activity (24). As shown in Figure 2, A and B, the acute-phase response in animals 8 h after IRI leads to recruitment of leukocytes in the lumen of the capillary blood vessels of the lumbar cord along with parenchymal infiltration compared with sham-operated controls. In Figure 2C, there were no leukocytes present in the lumen of the capillary vessels and spinal cord parenchyma 8 h after IRI in animals that were treated with epidural ICAM-1 Mab.
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Table 2 shows the paraplegia scores in relation to ICAM-1 treatment and the duration of ischemia. The objective of this phase of the study was to determine the critical ischemic duration in which the therapeutic efficacy of a neuroprotective drug can be optimally assessed. After 10 min of ischemic cross-clamp duration, no neurological deficits were present in either group. After 25 min of ischemic duration, complete paralysis was present in both groups. At ischemic aortic occlusion times between 15 and 20 min, variable grades of paraplegia scores ranging from 0 to 2 were elicited. Henceforth, we focused on the 15- to 20-min ischemic duration to assess the therapeutic effects of epidural ICAM-1 Mab on neurological function.
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Figure 3 shows the quantal response curves for animals that underwent spinal cord ischemia and were treated with epidural saline or ICAM-1 Mab. The quantal response curves, derived from the paraplegia scores in Table 2, represent the duration of aortic occlusion versus the probability of Grade 2 paraplegia. The ET50 in 24 animals treated with epidural saline was 15.4 ± 0.74 min (mean ± SE). The ET50 in the 24 animals treated with epidural ICAM-1 Mab was 22.1 ± 1.80 min. Both treatment group (P = 0.011) and time factors (P = 0.03) were significant, indicating that the probability of Grade 2 paraplegia increases over time and is diminished with treatment with epidural ICAM-1 Mab.
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Discussion |
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This study demonstrates that ICAM-1 expression in the capillary vessels of the lumbar spinal cord was increased in animals that were eight hours post-IRI after 30 minutes of aortic occlusion compared with sham-operated control animals. Similar findings by Hamada et al. (19) revealed that ICAM-1 messenger RNA expression was detectable three hours after injury and reached a maximum level in six hours in the thoracolumbar cord after traumatic spinal cord injury in rats. Although the traumatic spinal cord injury model used by Hamada et al. (19) is different from our IRI model, their findings are still relevant to the understanding of the ICAM-1 pathway in relation to spinal cord tissue damage, because the underlying mechanism of secondary injury is similar. Secondary injury processes after mechanical spinal cord trauma involve ischemia because of decreased blood flow and loss of microcirculation (25). Nevertheless, the increase of ICAM-1 expression in the vascular endothelial cells observed in this study provides corroborative evidence for the involvement of ICAM-1 in the pathophysiology of spinal cord ischemia.
The increase in ICAM-1 expression appears to be consistent with the histological/electron microscopic findings of leukocyte recruitment. Recruitment of leukocytes into the lumbar spinal cord capillaries, along with parenchymal infiltration, occurred within eight hours after IRI but was not observed in sham-operated control animals. These findings are also consistent with histological findings by Means and Anderson (26), who found that neutrophils appeared in the walls of vessels four hours after injury, leading to parenchymal infiltration after spinal cord tissue damage. Our finding that epidural ICAM-1 Mab ablated leukocyte recruitment eight hours after IRI further suggests that treatment modalities that block the ICAM-1 pathway may ameliorate leukocyte-mediated tissue injury.
As aforementioned, few studies have examined the role of the ICAM-1 pathway in relation to either traumatic or ischemic spinal cord injury (19,20). Hamada et al. (19) found that systemically administered ICAM-1 Mab diminished ICAM-1 messenger RNA expression and improved motor function in rats after traumatic spinal cord injury. Clark et al. (20) demonstrated that systemic ICAM-1 Mab reduced neurological deficits after ischemic spinal cord injury in rabbits. In this study, we report the administration of ICAM-1 Mab into the epidural space for neuroprotection after ischemic spinal cord injury. We found that epidural ICAM-1 Mab increased the ET50 or ischemic duration from 15.4 to 22.1 minutes. We chose the timepoint of 20 hours after ischemia to assess neurological function because the antileukocyte adhesion duration of action of ICAM-1 Mab is up to 20 hours (12,20). Consequently, we did not study time points well beyond 24 hours. Multiple dosing of the antibody may be needed to see a neuroprotective effect for a longer duration. Furthermore, this study was designed to evaluate the early events of the ICAM-1 pathway in the pathophysiology of ischemic injury.
In this study, the epidural route was chosen to develop an alternative strategy to inhibit leukocyte adhesion, with the objective of administering smaller doses of Mab as compared with systemic administration. The administration of ICAM-1 Mab for neuroprotection in humans has been severely limited by the occurrence of systemic immune reactions, suggesting that a revised dosing strategy may be needed to improve outcome in patients treated with antiadhesion drugs (15). Thus far, systemic doses of 1–2 mg/kg of ICAM-1 Mab have been used in experimental models of ischemic or traumatic CNS injury (18–20,26). Although we did not compare epidural versus systemic ICAM-1 Mab administration, our epidural dose of 0.5 mg/kg of ICAM-1 Mab is two- to fourfold less than previously used systemic doses. Further studies using smaller epidural doses in comparison with systemic doses are warranted to determine whether administering antileukocyte adhesion antibodies into the epidural space will confer neuroprotection while minimizing immunogenic side effects.
In conclusion, this study demonstrated the involvement of the ICAM-1 pathway in the pathophysiology of ischemic spinal cord injury after aortic occlusion in rabbits. Epidural ICAM-1 Mab ablated leukocyte recruitment and provided neuroprotection. Neuraxial administration of Mabs that inhibit leukocyte-mediated injury may be an alternative strategy to ameliorate spinal cord ischemia.
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Acknowledgments |
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Supported in part by the American Paraplegia Society, Jackson Heights, NY.
The authors thank Chris Jackson, BS, and Paige Preece, BS, University of South Florida Department of Anesthesiology, for their technical expertise in conducting this study.
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