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Anesth Analg 2001;92:1237-1244
© 2001 International Anesthesia Research Society

NEUROSURGICAL ANESTHESIA

The Effects of FK506 on Neurologic and Histopathologic Outcome After Transient Spinal Cord Ischemia Induced by Aortic Cross-Clamping in Rats

Loïc Lang-Lazdunski, MD, PhD*, Catherine Heurteaux, PhD{dagger}, Hervé Dupont, MD{ddagger}, Danielle Rouelle, BA{ddagger}, Catherine Widmann, BA{dagger}, and Jean Mantz, MD, PhD{ddagger}

*Department of Cardiovascular Surgery, Bichat University Hospital and Xavier Bichat Medical University, Paris, France; {dagger}Institute of Molecular and Cellular Pharmacology, Valbonne, France; and {ddagger}Department of Anesthesiology, Bichat University Hospital and Xavier Bichat Medical University, Paris, France

Address correspondence and reprint requests to Dr. Loïc Lang-Lazdunski, Service de Chirurgie Thoracique, Hôpital Européen Georges Pompidou, 20 Rue Leblanc, 75015 Paris, France. Address e-mail to loic.lang{at}wanadoo.fr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Spinal cord injury is a devastating complication of thoracoabdominal aortic surgery. We investigated the effect of the immunosuppressant FK506, a macrolide antibiotic demonstrated to have neuroprotective effects in cerebral ischemia models, in a rat model of transient spinal cord ischemia. Spinal cord ischemia was induced in anesthetized rats by using direct aortic arch plus left subclavian artery cross-clamping through a limited thoracotomy. Experimental groups were as follows: sham-operation; control, receiving only vehicle; FK506 A, receiving FK506 (1 mg/kg IV) before clamping; and FK506 B, receiving FK506 (1 mg/kg IV) at the onset of reperfusion. Neurologic status was assessed at 24 h and then daily up to 96 h with a 0 to 6 scale (0, normal function; 6, severe paraplegia). Rats were randomly killed at 24, 48, or 96 h, and spinal cords were harvested for histopathology. Physiologic variables did not differ significantly among experimental groups. All control rats suffered severe and definitive paraplegia. FK506-treated rats had significantly better neurologic outcome compared with control. Histopathologic analysis disclosed severe injury in the lumbar gray matter of all control rats, whereas most FK506-treated rats had less injury. These data suggest that FK506 can improve neurologic recovery and attenuate spinal cord injury induced by transient thoracic aortic cross-clamping.

Implications: A single dose-injection of the immunosuppressant FK506 significantly improved neurologic outcome and attenuated spinal cord injury induced by transient thoracic aortic cross-clamping in the rat.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Spinal cord ischemia with resulting paraplegia is a devastating complication after repair or replacement of the thoracoabdominal aorta (1,2). Increasing spinal cord tolerance to ischemia by injecting neuroprotective drugs before aortic clamping, or reducing spinal cord reperfusion injuries, would represent an important advance (1). FK506, or tacrolimus (Fujisawa Pharmaceuticals, Osaka, Japan), a macrolide antibiotic with major immunosuppressive properties, is used to prevent rejection after organ transplantation (3). Several studies have reported a neuroprotective action of FK506 in experimental models of cerebral ischemia or spinal cord injury (4–8). The neuroprotective effect of FK506 is thought to be related mainly to the inhibition of the protein phosphatase calcineurin, but FK506 might also be neuroprotective through a reduction of peroxynitrite radical production and through an inhibition of Ca2+-triggered apoptosis (4,5).

We designed this study to determine whether FK506 could improve neurologic outcome and reduce histologic evidence of spinal cord injury in a rat model of transient spinal cord ischemia.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Sprague-Dawley male rats weighing 350–375 g were used in this study. All animals were allowed free access to laboratory chow and tap water in day/night regulated quarters at 25°C. Animal care and experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (9) and were approved by the local Animal Studies Committee.

Anesthesia was induced in a chamber containing 3% halothane and was maintained by inhalation through a snout mask of 1.5% halothane in balance oxygen. Rectal temperature was continuously monitored with a flexible probe inserted 5 cm into the rectum in all animals, and it was maintained at 37.5°C ± 0.5°C by a thermal pad and a heating lamp. Rats were placed in the supine position, and a longitudinal incision was made through the skin in the sternal region. The left jugular vein was used for IV injection of drugs or vehicle. The chest wall was incised from the apex of the manubrium caudad along the left sternal border, to the third rib. The thymus was retracted superiorly, and the aortic arch was isolated between the left common carotid and the left subclavian arteries. Spinal cord ischemia was induced by aortic arch plus left subclavian artery cross-clamping for 14 min by use of two Micro Vessel Clips (catalog no. 14-1020; Biomedical Research Instruments, Rockville, MD). Then the clips were removed and the wound was closed in layers. Sham-operated animals underwent an identical surgical procedure except for aortic arch and left subclavian artery clamping. Animals were allowed to recover in a plastic box at 28°C for 3 h and were then placed in their cages with free access to food and water. The Credé maneuver was used twice daily to empty the urinary bladders of paraplegic animals.

Animals were randomly assigned to different study groups as follows: in the Sham-Operation group (n = 15), all surgical procedures were performed by using similar conditions except for aortic and subclavian artery clamping; in the Control group (n = 20), animals received only vehicle IV; in the FK506 A group (n = 20), FK506 (1 mg/kg) was injected IV 30 min before aortic clamping; and in the FK506 B group (n = 20), FK506 (1 mg/kg) was injected IV at the onset of reperfusion.

All rats received a similar volume of solutions: 1 mL IV through the internal jugular vein during surgery. FK506 was dissolved in 10% ethanol in 1 mL of saline.

Separate groups of animals were anesthetized by using similar conditions: Sham-Operation (n = 3), Control (n = 5), FK506 A (n = 5), and FK506 B (n = 5). An IV catheter (24-gauge) was inserted in the tail artery and connected via a transducer to a pressure monitor (Hewlett-Packard, Palo Alto, CA) for continuous monitoring of distal mean arterial blood pressure. Mean arterial blood pressure was monitored throughout the procedure. The catheter was removed after 30 min of reperfusion, and the tail incision was closed. Arterial blood gases were measured 10 min before aortic clamping and after 10 min of reperfusion in samples obtained from the tail artery with a blood gas and pH analyzer (Ciba-Corning Diagnostics, East Walpole, MA). Blood glucose levels were determined in samples obtained from the jugular vein just before aortic clamping (Accu-Chek Easy Blood Glucose Monitor; Boehringer Mannheim, Indianapolis, IN).

Serial assessments of motor and sensory functions in the hind limbs of all rats were performed at 24 h and then daily up to 96 h of reperfusion by using the criteria of Le May et al. (10) with modifications:

(a) Walking with lower extremities
  1. 0. Normal.
  2. 1. Toes flat under body when walking, but ataxia is present.
  3. 2. Knuckle walking.
  4. 3. Movements in lower extremities but unable to knuckle walk.
  5. 4. No movement, drags lower extremities.

(b) Pain sensation
  1. 0. Normal, withdrawal to toe pinch.
  2. 1. Squeals to toe pinch but does not withdraw.
  3. 2. No reaction to toe pinch.

A motor sensory deficit index (MSDI) was calculated for each animal at each time point. The final index was the sum of the scores (a) and (b), and the maximum deficit was indicated by a score of 6.

Animals were randomly assigned to euthanasia at 24, 48, or 96 h of reperfusion: Sham-Operation group, n = 5 for each time; for the Control, FK506 A, and FK506 B groups, n = 5 at 24 h, n = 5 at 48 h, and n = 10 at 96 h. Animals were anesthetized with 3% halothane and transcardially perfused with 100 mL of 0.9% saline solution at 4°C. Spinal cords were quickly harvested and either placed in fresh 4% paraformaldehyde at 4°C for 48 h or fresh frozen in isopentane on dry ice and stored at -70°C.

Spinal cords were removed from 4% paraformaldehyde after 48 h fixation. Specimens were dehydrated in alcohol 95% for 30 min, followed by four changes of 100% alcohol for 1 h each and five changes of toluene for 1 h each under vacuum at 37°C. Spinal cords were infiltrated with paraffin and embedded in paraffin at 57°C under vacuum and pressure. Transverse sections were made on a microtome (Leica, Rueil-Malmaison, France). Ten-micrometer sections were obtained through the lumbar spinal cord. Sections were stained with hematoxylin and eosin (H&E) and Luxol fast blue staining method and were examined under the light microscope. All animals had their spinal cords examined. Five representative sections taken from the L1 to L5 segments were examined in each animal. The neuropathologist who performed the examination was blinded to the experimental conditions and coded the section with maximum gray matter damage in each rat.

DNA nick-end labeling by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) method was used to search for morphologic features of apoptosis. Coronal 10-µm frozen sections were used and processed according to the TUNEL method described by Gavrieli et al. (11) by using the in situ cell death detection kit (Hoffmann-La Roche, Ltd., Basel, Switzerland) and the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). Cells with nuclei well-stained by the TUNEL method or containing apoptotic bodies were considered to be apoptotic. Some neurons had very faint staining and did not contain apoptotic bodies. Those neurons were considered to be necrotic. Necrotic and apoptotic neurons were counted in the H&E and TUNEL sections. Surviving motor neurons were also counted in all spinal cord sections.

The Rexed classification was used to describe the locations of damaged neurons in gray matter (12). The degree of gray matter ischemic damage was judged individually for each of three regions as reported by Taira and Marsala (13): dorsal horn, damage involving laminae I to VI; intermediate gray matter, damage involving laminae VII and X; and ventral horn, damage involving laminae VIII and IX. Each region was scored according to the following criteria: 0, normal histopathology, no detectable changes; 1, damage affecting <10% of the scored area; 2, damage affecting 10% to 50% of the scored area; and 3, damage affecting more than 50% of the scored area. The final neuropathologic score for each spinal cord ranged between 0 (normal spinal cord) and 9 (more than 50% postischemic damage).

Statistical analyses of measured physiologic data were performed by a repeated-measures analysis of variance. All physiologic data are expressed as mean ± SD. Neurological scores were analyzed with Kruskal-Wallis tests, followed by Mann-Whitney U-tests when significant. Differences in the numbers of neurons between groups were assessed by Student’s t-test. Differences were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Tables 1 and 2 present the physiologic variables recorded in animals during the experiment. There was no significant difference among experimental groups at any time.


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  		Table 1. Physiologic Variables
 

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  		Table 2. Blood Gases and Glucose Levels
 
Three rats (15%) in the Control group died either during or after the operation, and one rat in the FK506 B group died after surgery. Those rats were replaced by other rats to reach the appropriate n in each group. Table 3 presents neurologic outcome up to 96 h. All sham-operated rats had a normal postoperative neurologic outcome, whereas all control rats had an MSDI of 6 at 24 h and remained severely paraplegic throughout the observation period, with no improvement in either sensory or motor function. Neurologic outcome was significantly better in FK506-treated rats at any time considered (Table 3). All FK506-treated rats had some form of flaccid paraplegia after recovery from anesthesia, but they began to recover motor function between 3 and 6 h after surgery. At 24 h, 50% of FK506-pretreated rats and 45% of FK506-posttreated rats suffered mild to moderate deficit (MSDI 2–4), whereas the remaining FK506-treated rats in both groups suffered severe deficit (MSDI 5–6) (Table 3). Two FK506-treated rats in each group had a worsening neurologic status between 24 and 48 h (10% in each group), but none had worsening neurologic status after 48 h. FK506-treated rats with an MSDI of 6 usually had spastic paraplegia persisting until euthanasia.


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  		Table 3. Neurologic Status (Motor Sensory Deficit Index [MSDI]) at 24, 48, and 96 h of Reperfusion
 
H&E and Luxol fast blue staining were used to analyze neuronal cell death in gray matter ( Fig. 1). The extent of ischemic damage was grossly proportional to the neuropathologic score. Ischemic damage was observed almost exclusively in gray matter, which contained typically necrotic neurons with eosinophilic cytoplasm (red neurons) and loss of cytoplasmic structures (ghost neurons), but also neurons demonstrating apoptotic features.



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  		Figure 1. Histopathology. Luxol fast blue staining of transverse spinal cord sections. Bar in panel E represents 500 µm and applies to panels A and C. B, D, and F are higher-magnification photomicrographs of the area enclosed within the boxes in A, C, and E, respectively. Bar in F represents 130 µm and also applies to B and D. Panels A and B, specimens obtained from an animal in the Sham-Operation group killed at 96 h with a motor sensory deficit index (MSDI) of 0. The gray matter architecture was preserved with normal motor neurons in the ventral horn. Panels C and D, specimens obtained from an animal in the Control group killed at 96 h with an MSDI of 6 and severe flaccid paraplegia. The gray matter architecture was destroyed by the insult. Coalescent cavities and a prominent inflammatory cell infiltrate may be seen within the infarct that is well demarcated from surrounding tissue (Panel C). The infarct did not extend into the white matter (panel C). There is neuronal vacuolization, and most large {alpha} motor neurons did not survive the ischemic insult (Panel D). Panels E and F, specimens obtained from an animal in the FK506 A group killed at 96 h with an MSDI of 3. The gray matter architecture is globally conserved, and most large {alpha} motor neurons appear to have survived the ischemic insult. There is no leukocytic infiltration in the ventral horn (Panel E).

 
Sham-operated rats had normal spinal cords, with the neuropathologic score being 0 ± 0 for this group. All control rats had very severe ischemic injury in gray matter, with extended infarcts involving laminae II to X and containing neutrophils and mononuclear phagocyte infiltrates. Only a few motor neurons appeared to have survived the ischemic insult. In the most severe cases, white matter surrounding gray matter was also damaged in spinal cord sections of rats killed at 48 h of reperfusion or later. Fifty percent of FK506-pretreated rats and 40% of FK506 posttreated rats had mild to moderate neuronal damage in gray matter, with mild vacuolization of gray matter predominantly in laminae III to VII. Laminae I and II in dorsal horns and lamina X around the ependymal canal were usually preserved. Lamina VIII and Lamina IX containing motor neurons were usually spared. Variable-sized confluent necrotic areas were seen predominantly in laminae III to VII of animals with an MSDI of 5 or 6. The infarct areas had variable size and distribution and were characterized by destruction of the normal gray matter architecture. Those infarcts did not involve laminae I and II in dorsal horns and laminae VIII to X in animals with an MSDI of 5. Those infarcts were larger in animals with an MSDI of 6 and extended to laminae VIII, IX, and X. The number of intact motor neurons per section was significantly more in FK506-treated animals ( Table 4).


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  		Table 4. Effect of FK506 Treatment on the Survival of Large Motor Neurons
 
The histopathologic changes in FK506-treated animals were milder than those observed in the Control group and occurred with mild to moderate neutrophilic infiltration in the gray matter, compared with major neutrophilic infiltration in the Control group ( Fig. 2). The mean neuropathologic score for the Control group was 8.3 ± 0.7, and the mean neuropathologic scores were 5.8 ± 1.9 (range, 2 to 8) and 6.2 ± 1.5 (range, 3 to 8) for the FK506 A and FK506 B groups, respectively.



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  		Figure 2. In situ DNA nick end labeling (TUNEL) staining. Representative section obtained from the lumbar spinal cord of an FK506-treated rat killed after 24 h of reperfusion with mild motor deficit. There are numerous TUNEL-positive neurons scattered within the gray matter, especially in the dorsal horns. Mostly small-sized neurons are labeled by the TUNEL method. There is no TUNEL-positive motor neuron in the ventral horns.

 
Apoptosis characterized by chromatin condensation, DNA fragmentation, nuclear shrinkage, and fragmentation of nuclear bodies (apoptotic bodies) was visualized by the in situ TUNEL method. Sham-operated animals had no TUNEL labeling in the spinal cord. Control animals had numerous TUNEL-positive nuclei diffusely distributed within the gray matter at 24 and 48 h, and much less at 96 h, of reperfusion. Most TUNEL-positive neurons were located in the dorsal horns and intermediate gray matter, but also to a lesser extent in the ventral horns. The numbers of apoptotic neurons increased in the ventral to dorsal direction, with the maximum in laminae I and II ( Fig. 3). In sections from FK506-treated rats, TUNEL-positive cells were distributed in a similar way within the gray matter. However, the numbers of TUNEL-positive cells were significantly decreased in FK506-treated rats after 24 h of reperfusion ( Fig. 4).



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  		Figure 3. Distribution of the necrotic neurons in gray matter of the lumbar spinal cord of animals killed after 24 h of reperfusion. Reported are all neurons counted on a high-power field. The difference between the Control and the FK506 A or FK506 B groups is not statistically significant in any area.

 


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  		Figure 4. Distribution of the apoptotic neurons in they gray matter of the lumbar spinal cord of animals killed after 24 h of reperfusion. Reported are all neurons counted on a high-power field. The difference between the Control and FK506 A or FK506 B groups is statistically significant in all three areas (dorsal horn, 69 ± 6 vs 23 ± 12 or 18 ± 10; intermediate zone, 27 ± 6 vs 10 ± 4 or 6 ± 5; ventral horn, 11 ± 3 vs 4 ± 2 or 3 ± 2).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Paraplegia resulting from spinal cord ischemia is a devastating complication of thoracoabdominal aortic surgery (1,2). Despite various surgical adjuncts and pharmacological interventions, spinal cord ischemic injury follows aortic repair or replacement in 0% to 21% of patients (1,2). Numerous animal models have been developed to study the pathophysiologic mechanisms leading to irreversible spinal cord ischemic injury and also to test neuroprotective drugs (10,14). The rat model used in this study has been adapted from other models that use transient cross-clamping of either the aortic arch or the aortic arch and the subclavian arteries (10,15). This model is highly reproducible because control rats operated in strict normothermia and clamped for 14 min usually suffer severe and definitive paraplegia (16). Operative mortality was low (1%), and severe bowel ischemia occurred in only 4% of animals. Spinal cord arterial vascularization is almost similar in rats and humans, with few heterosegmental anterior radicular arteries reaching the anterior spinal artery and one major radicular artery supplying the lower thoracic and lumbar spinal cord (17). Combined clamping of both the aortic arch and the left subclavian artery reproduces the operative situation encountered during repair of the descending thoracic or thoracoabdominal aorta in humans. Blood flow in the spinal cord, liver, bowel, and kidneys is dramatically reduced during the clamping period (14). This usually results in metabolic acidosis, a factor that increases neurologic injury (18).

There is growing enthusiasm regarding the neuroprotective effects of FK506. This drug has an excellent diffusion across the blood-brain barrier and is neuroprotective at doses of 0.1 to 1 mg/kg (4–6). The blood level of the drug is maximal 15–30 minutes after IV injection, and the brain level of FK506 increases rapidly after IV injection and remains at a constant level from 15 minutes after the injection to 72 hours later (4). FK506 has demonstrated neuroprotective effects in several models of focal and global cerebral ischemia (4–7).

The precise mechanism of action of this drug is not completely understood, and its neuroprotective action is probably multifactorial (5). FK506 complexes the 12-kilodalton immunophilin FKBP12 (FK binding protein 12), resulting in inhibition of the Ca2+/calmodulin-dependent protein phosphatase calcineurin (4,5). It has been suggested that the neuroprotective effect of FK506 may be mediated by suppression of the NO synthase activation that occurs after ischemic depolarization and subsequent NO production during ischemia and early reperfusion. However, a recent study (5) demonstrated that FK506 does not attenuate ischemia-evoked NO production. FK506 might also prevent secondary deterioration of mitochondrial function and apoptosis signaling after cerebral ischemia (19,20). FK506 attenuates leukocyte rolling and subsequent accumulation of leukocyte in the retina after transient ischemia and reperfusion (21). In addition, FK506 inhibits superoxide radical production in neutrophils and suppresses the production of inducible NO synthase in cultured macrophages (22,23). Both inducible NO synthase and superoxide radicals have been implicated in the pathophysiology of postischemic central nervous system damage (24). Thus, FK506 may act by attenuating white cell-mediated postischemic processes in the spinal cord. Finally, FK506 has been reported to be neurotrophic, with a potency comparable to those of well known neurotrophic proteins such as BDNF, NT3, GDNF, and NGF, and it improves functional recovery after spinal cord injury (8,25).

In this study, treatment of rats with FK506 significantly improved neurologic outcome but incompletely prevented gray matter injury despite adequate dose injection. However, this study demonstrates that a single injection of FK506 before or even after aortic clamping can attenuate significantly the apoptotic phenomena in the gray matter of most treated animals. FK506 might have attenuated ischemia/reperfusion injury in the spinal cord because rats in Group B did receive the drug at the onset of reperfusion and generally had a better neurologic outcome than control rats. Furthermore, FK506-treated rats had minimal to moderate neutrophilic infiltration in the gray matter compared with control animals, suggesting a direct action of FK506 on neutrophils. Our data are in accordance with other studies that reported a sustained neuroprotective effect for 72 hours after a single-dose injection of FK506 (4,7). It should be pointed out that the ischemic insult used in this study was severe; 100% of control rats suffered severe and definitive flaccid paraplegia and demonstrated extensive spinal cord injury. Therefore, it can be postulated that the neuroprotective effect of FK506 could have been more evident with a shorter ischemic period (e.g., 11–12 minutes).

FK506 reduces infarct volume by 50%–60% in transient focal cerebral ischemia models (4,5). However, this drug seems to be less efficient in models of transient global cerebral ischemia. Thus, studies in gerbils demonstrated that FK506 rescued only 25%–50% of hippocampal neurons after five minutes of global cerebral ischemia (6,7). In this study, FK506 rescued grossly 25%–50% of neurons in gray matter. Neither necrotic nor apoptotic phenomena were prevented by FK506 in this model. However, those processes were found attenuated in most treated animals. In this study, the effectiveness of FK506 was tested both in a pretreatment and in a posttreatment regimen. Because this study aimed to investigate preventive treatment that may prevent spinal cord injury sustained mostly during elective aortic surgery, an effective pretreatment regimen seems clinically more relevant than a posttreatment one. However, posttreatment with FK506 might still be useful in patients with delayed neurologic deficit or in patients with ruptured aortic aneurysm and neurologic deficit in evolution, to attenuate spinal cord injury.

Modifications of arterial blood pressure and of body temperature have been reported by some investigators in animals receiving FK506 IV (6). However, hypothermia cannot account for the neuroprotective effects of FK506 observed in this study because rectal temperature was closely monitored in all rats and did not differ among experimental groups. In addition, no significant difference was observed with either blood glucose or arterial blood pressure among experimental groups used for monitoring physiologic variables, and these are two factor that may influence the level of spinal cord injury (10,13).

Major adverse effects have been observed with the chronic use of FK506. These include drug-induced neurotoxicity varying from 5% to 30% of patients and consisting in severe organic brain syndrome, as well as aphasia, ataxia, seizures, coma, and leukoencephalopathy (3). However, it seems improbable to observe such side effects with a single-dose injection of FK506.

Now only a few drugs are candidates for pilot human studies in the field of thoracoabdominal aortic surgery. Riluzole, memantine, and magnesium sulfate are antiexcitotoxic drugs currently in use among patients with various neurologic disorders, and they all have demonstrated neuroprotective effects in spinal cord ischemia models (26,27). FK506 may also be a candidate because it has been used for several years after organ transplantation (3). In addition, this drug may be of special interest in the setting of thoracoabdominal aortic surgery because it also protects the kidney, liver, and bowel against ischemia/reperfusion injury (28–30). Further research in larger animal species is warranted to confirm that this drug can reliably ameliorate the spinal cord injury induced by aortic cross-clamping.

In conclusion, these data suggest that a single-dose injection of FK506 can significantly improve neurologic outcome and reduce neurologic injury after transient spinal cord ischemia induced by thoracic aortic cross-clamping in the rat. Further studies are warranted to confirm that FK506 may be useful to extend spinal cord protection during high-risk surgical procedures on the descending thoracic or thoracoabdominal aorta.


   Acknowledgments
 
Supported, in part, by le Centre National de la Recherche Scientifique.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication January 17, 2001.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press