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Anesth Analg 2003;96:839-846
© 2003 International Anesthesia Research Society

NEUROSURGICAL ANESTHESIA

Lack of Evidence for Apoptosis as a Cause of Delayed Onset Paraplegia After Spinal Cord Ischemia in Rabbits

Takashi Kiyoshima, MD, Shiro Fukuda, MD, Mishiya Matsumoto, MD, Yasuhiko Iida, MD, Satoe Oka, MD, Kazuhiko Nakakimura, MD, and Takefumi Sakabe, MD

Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, Japan

Address correspondence and reprint requests to Takefumi Sakabe, MD, Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, 1–1-1 Minami-Kogushi, Ube, Yamaguchi 755–8505, Japan. Address e-mail to sakabe{at}yamaguchi-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms for delayed onset paraplegia after transient spinal cord ischemia are not fully understood. We investigated whether apoptotic motor neuron death is involved in its development. Spinal cord ischemia was induced for 15 min by occlusion of the abdominal aorta in rabbits. At 8, 24, or 48 h after reperfusion, hind limb motor function was assessed, and the lumbar spinal cord was examined morphologically (hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining) and biochemically (breakdown products of {alpha}-fodrin and patterns of DNA changes). At each time point, 14 rabbits were studied (7 for histopathology and 7 for biochemical analysis). Six rabbits served as sham controls. Delayed motor dysfunction developed in two thirds of the rabbits. The motor neurons in the rabbits with motor dysfunction (not paraplegia) showed swelling and a finely granular dispersed Nissl substance. In paraplegic rabbits, destruction of the gray matter and prominent inflammatory cell infiltration were observed. No apoptotic motor neuron was found in any rabbit. There was neither detectable increase in a caspase-3-mediated breakdown product of {alpha}-fodrin, nor DNA laddering in any rabbit. The results suggest that apoptosis has a negligible role in the pathophysiology of delayed paraplegia in the spinal cord ischemia model examined.

IMPLICATIONS: Although the possibility of apoptotic motor neuron death cannot be completely excluded, delayed onset paraplegia after transient spinal cord ischemia is largely associated with necrotic cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Preventing ischemic paraplegia after thoracoabdominal aortic aneurysmal surgery is a challenging issue for both anesthesiologists and surgeons. There seem to be at least two types of paraplegia: acute and delayed. Acute paraplegia may be defined as presenting at emergence from anesthesia. In contrast, delayed paraplegia typically develops within a few days without a manifestation early after the operation. These two types of paraplegia are also observed in well-characterized animal models of transient spinal cord ischemia (1–5).

It is difficult to treat acute paraplegia once it occurs. However, in the case of delayed paraplegia, there might be a therapeutic time window. It has been suggested that destruction of the blood-spinal cord-barrier and subsequent spinal cord edema that occurs several hours after reperfusion may contribute to delayed paraplegia (6). Apoptosis of motor neurons has been reported after transient spinal cord ischemia (7–9). Sakurai et al. (8) observed apoptotic changes of motor neurons with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) staining and DNA laddering 48 h after 15 min of ischemia in rabbits and suggested that apoptosis plays an important role in delayed paraplegia. In our previous studies, we observed delayed paraplegia in many rabbits subjected to 20–25 min of ischemia (3–5). However, histological examination (48 h after reperfusion) in rabbits with delayed paraplegia consistently revealed a destruction of the gray matter and prominent inflammatory cell infiltration. These morphological changes are distinct from apoptotic cell death (10). Therefore, it is still a controversial issue whether apoptosis is the cause of delayed paraplegia after transient spinal cord ischemia.

In the present study, we sought to determine morphologically and biochemically at 3 time points (8, 24, and 48 h after reperfusion) whether apoptosis of motor neurons in the spinal cord occurs after transient spinal cord ischemia and whether apoptosis plays an important role in the development of delayed paraplegia. We chose 15 min of ischemia so as to have a chance, if any, to observe apoptotic changes of motor neurons and to compare our results with those of Sakurai’s group (8,9,11).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The protocol of this study was approved by the Ethics Committee for Animal Experiment at Yamaguchi University School of Medicine. Forty-eight New Zealand white rabbits weighing 2.9 ± 0.4 (mean ± SD) kg were used in this study. The methods for surgical preparation producing spinal cord ischemia, postischemic management, and neurological and histopathological (hematoxylin and eosin [HE] staining) evaluation were almost the same as our previous studies (3–5).

The rabbits were anesthetized with 2%–3% isoflurane in 40% oxygen/60% nitrogen. The lungs were mechanically ventilated. Temperatures were monitored with a calibrated esophageal thermistor and needle-type thermistor inserted into the paravertebral muscle at the level of L4-5. The paravertebral muscle temperature was controlled at {approx}38.0°C throughout the study. PE-60 catheters were inserted into both femoral arteries to measure blood pressure above and below the aortic occlusion.

In the right lateral decubitus position, a skin incision was made at the 12th costal level. A PE-60 catheter was retroperitoneally placed around the aorta immediately distal to the left renal artery to produce spinal cord ischemia.

Segmental spinal cord evoked potentials (SSCEPs) were recorded in a bipolar fashion (L5 and L6). SSCEPs were recorded every 2 min until 15 min after reperfusion. Because poor recovery ratio of the amplitude of the third negative wave (N3) to the first negative wave (N1) (N3/N1) predicts poor outcome after reperfusion (12), we calculated N3/N1 at 15 min after reperfusion to predict the final neurologic outcome (48 h after reperfusion) in the current study.

The rabbits were randomly assigned to one of four groups depending on the time schedule of the final evaluation after ischemia or sham procedure: 8 h after ischemia (8-h group; n = 14), 24 h after ischemia (24-h group; n = 14), 48 h after ischemia (48-h group; n = 14), and sham operation (control group; n = 6). All groups were further divided into 2 groups (n = 7 in ischemia groups and n = 3 in control group), each of which was used for either histological evaluation or biochemical analysis of the spinal cord.

After completion of surgery, end-tidal isoflurane concentration was maintained at 2%. Spinal cord ischemia was produced for 15 min. In the control group, only surgical manipulation without aortic occlusion was performed. After final recording of SSCEPs (15 min after reperfusion), all incisions were sutured. Isoflurane was discontinued, and the lungs were ventilated with 100% oxygen. Extubation of the trachea was performed when adequate spontaneous ventilation occurred. The rabbits were allowed to recover, and an antibiotic (cephazolin 30 mg/kg IM) was administered once daily.

At 8, 24, and 48 h after reperfusion, the rabbits were neurologically assessed by an observer unaware of the treatment group using the five-point grading scale proposed by Drummond and Moore (13): 4 = normal motor function; 3 = ability to draw legs under body and hop but not normally; 2 = some lower-extremity function with good antigravity strength but inability to draw legs under body; 1 = poor lower-extremity motor function and weak antigravity movement only; 0 = paraplegic with no lower-extremity function.

After completion of the scoring of neurologic function at scheduled time (8, 24, or 48 h, n = 7 each; 8 h for control, n = 3), the animals were reanesthetized, and trans-cardiac perfusion and fixation were performed with 500 mL of 10% phosphate-buffered formalin. The coronal sections of the spinal cord (8 µm) at the level of L5 were stained with either HE or TUNEL staining, and the latter was performed to detect double-strand breaks in genomic DNA using a kit (Apoptosis in situ Detection Kit, WAKO Jun-yaku, Tokyo, Japan) according to the manufacturer’s specifications with modifications. The staining technique was verified using spinal cord samples treated with deoxyribonuclease as well as brain samples obtained after transient forebrain ischemia and reperfusion. Histopathological evaluation was performed with two sections for each rabbit by an observer unaware of the treatment groups. In TUNEL staining, we differentiated between apoptotic neurons and necrotic neurons by the following characteristics: the apoptotic neurons show the nucleus stained dark brown with or without chromatin condensation and apoptotic bodies in association with translucent cytoplasm, whereas necrotic neurons, if stained brown, show a diffuse light brown staining not only in the cell nucleus, but also in cytoplasm (14).

For biochemical analysis, the rabbits were reanesthetized with 2% isoflurane in oxygen at scheduled times (8, 24, or 48 h, n = 7 each; 8 h for control, n = 3). The spinal cords were quickly removed immediately after they were killed using the plunger of a 1-mL syringe, frozen in liquid nitrogen, and then stored at -80°C.

For analysis of {alpha}-fodrin and its breakdown products, the frozen spinal cord was homogenized, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed according to the method previously reported (15). The antigen-antibody complex was visualized by the enhanced chemiluminescence western blotting detection kit. The proteolytic fragments of {alpha}-fodrin were quantified by an image analyzer (Densitograph AE-6905C, Atto, Tokyo, Japan). Protein concentration was measured according to the method of Lowry et al. (16) using bovine serum albumin as a standard. Antibody to rabbit {alpha}-fodrin was purchased from Affiniti (Exeter, United Kingdom), the molecular weight standard from Bio-Rad (Richmond, CA), and the enhanced chemiluminescence western blotting detection kit from Amersham International (Buckinghamshire, United Kingdom).

For analysis of DNA fragmentation by electrophoresis, the frozen spinal cord (-80°C) was homogenized in the enzyme reaction solution provided in a kit (Apoptosis Ladder Detection Kit, Wako) with the same procedure as the immunoblotting. DNA was extracted from the homogenate with use of the same kit according to the manufacturer’s specifications with modifications. Then, extracted DNA was separated on a 1.5% agarose gel by electrophoresis, visualized with ethidium bromide, and photographed under UV illumination.

Parametric data are presented as mean ± SD. The physiologic variables were analyzed by a repeated-measures analysis of variance. Where differences were identified, Scheffé post hoc test for intergroup comparisons was performed. The correlation of neurologic function scores of hind limb and N3/N1 recovery in SSCEP at 15 min after reperfusion and the correlation of neurologic function scores of hind limb and the amount of {alpha}-fodrin fragments were analyzed by Spearman rank correlation. P < 0.05 was evaluated as statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
There were no significant differences in physiological values among the groups except for distal mean arterial blood pressures during ischemia, which were significantly lower in the ischemia groups than those of the control group (Table). All rabbits survived until the prescheduled time period for neurologic assessment and other evaluations.


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  		Table 1. Physiologic Variables
 
Delayed motor dysfunction was observed in two thirds of the rabbits. Delayed paraplegia developed in 6 of 28 rabbits at 24 to 48 h after ischemia (Fig. 1). In the 48-h group, evaluation of the relationship between the final neurologic outcome and the N3/N1 recovery rate in SSCEP at early stage (15 min after reperfusion) revealed a significant correlation (r = 0.793, P < 0.01). No rabbits with the poor SSCEP recovery (N3/N1 <50%) were able to hop 48 h after reperfusion (score <= 2). All rabbits in the control group showed normal motor function (score 4, not shown in Fig. 1).



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  		Figure 1. Changes of individual neurologic function scores after 15 min of ischemia in 3 groups (8-, 24-, and 48-h groups). Each symbol represents data for one rabbit. Neurologic function scores range from 0 (paraplegia) to 4 (normal). The segmental spinal cord evoked potential (SSCEP) recovery rate is the ratio of the amplitude of the third negative wave (N3) to the first negative wave (N1) (N3/N1) in percent at 15 min after reperfusion.

 
In HE staining, motor neurons in the rabbits with normal motor function (score 4) 8 h after reperfusion showed either completely normal appearance (Fig. 2A) or swelling of the cell body and finely granular dispersed Nissl substance without apparent changes of the nucleus or nucleolus (Fig. 2B), the latter changes seen in the rabbits with a poor SSCEP recovery. In the rabbits that exhibited moderate motor dysfunction (score 2), most motor neurons showed similar abnormality (Fig. 2C). At 24 or 48 h after reperfusion, in the rabbits developing motor dysfunction (score 3), most motor neurons (Fig. 2E) seemed essentially similar to those in Figure 2C. In the rabbits with paraplegia, the structure of the spinal cord gray matter was destroyed, and most motor neurons disappeared (Fig. 2, F and I). A prominent inflammatory cell infiltration can be seen in Figure 2I. The nucleolus of the motor neuron was present until the late stage of degeneration.



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  		Figure 2. Microphotographs of the sections showing motor neurons in the spinal cord (L5) stained with hematoxylin-eosin. (A–C) Eight-hour group (8 h after reperfusion). (D–F) Twenty-four-hour group (24 h after reperfusion). (G–I) Forty-eight-hour group (48 h after reperfusion). (A) Representative section from a rabbit with normal motor function (score 4) and a good segmental spinal cord evoked potential (SSCEP) recovery. Normal appearance of the motor neuron can be seen. (B) Section from a rabbit with normal motor function and a poor SSCEP recovery. Swelling of the cell body and finely granular dispersed Nissl substance can be seen. These appearances were observed in some motor neurons. (C) Section from a rabbit with moderate motor dysfunction (score 2). Most of the motor neurons showed almost the same abnormality as that in Figure 2B. (D) Normal appearance of the motor neuron from the rabbit with normal motor function and a good SSCEP recovery. (E) Section from a rabbit with mild motor dysfunction (score 3). Most motor neurons seemed essentially similar to those in Figure 2C. The nucleolus can clearly be seen. (F) Section from a rabbit with paraplegia (score 0). The motor neuron is shrunken and Nissl substance is lost. The nucleus is also shrunken. Inflammatory cells are seen. (G) Normal appearance of the motor neuron from a rabbit with normal motor function and a good SSCEP recovery. (H) Section from a rabbit with mild motor dysfunction (score 3). The motor neuron is shrunken, but the nucleolus is still seen. (I) Section from a rabbit with paraplegia (score 0). The structure of the gray matter is destroyed. The motor neuron is shrunken, and no Nissl substance is demonstrable. The cytoplasm is uniformly structureless. A prominent inflammatory cell infiltration can be seen.

 
Most motor neurons under degeneration showed no brownish nuclei with TUNEL staining (Fig. 3C). There were a few brownish-stained motor neurons in 3 rabbits 48 h after reperfusion, but they showed diffuse brown staining in both the cell nucleus and cytoplasm (Fig. 3D), the characteristics not being consistent with apoptosis. In laminae V-VII, where the destruction of the gray matter was detected by HE staining, there were a few neurons (not motor neurons) (<5%) with their nuclei stained brown in 6 of 14 rabbits at 24 and 48 h after reperfusion.



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  		Figure 3. Microphotographs of sections showing motor neurons in the spinal cord (L5) stained with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) method. (A) A positive control slide (a sample from normal spinal cord treated with deoxyribonuclease). A motor neuron showing the brownish nucleus is present. (B) A neuron showing the brownish nucleus with translucent cytoplasm in laminae V-VII in a rabbit with normal motor function (24 h after reperfusion). (C) A degenerated motor neuron in a rabbit with moderate motor dysfunction (48 h after reperfusion). The nucleus is not stained brown. (D) A motor neuron in a rabbit with moderate motor dysfunction (48 h after reperfusion). The motor neuron shows a diffuse light brown staining not only in the cell nucleus, but also in cytoplasm.

 
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting results were as follows: At 8 h after reperfusion, 145- and 150-kDa fragments generated from {alpha}-fodrin (calpain-mediated breakdown products) were detected but only in a small amount. The amount of 145- and 150-kDa fragments increased in the rabbits with poor motor function 48 h after reperfusion (Fig. 4). However, there was no significant increase in a 120-kDa fragment, which is a caspase-3-mediated breakdown product of {alpha}-fodrin (17). Regardless of the time of evaluation, the rabbits with poor motor function had a larger amount of {alpha}-fodrin fragments (145 and 150 kDa), and the amount of the fragments increased according to worsening of the neurologic score (r = 0.838, P < 0.001; Fig. 5).



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  		Figure 4. Immunoblots showing the fragments of {alpha}-fodrin 48 h after reperfusion. The numbers below the blots indicate the neurologic function score (C, control). The amount of 145- and 150-kDa fragments of {alpha}-fodrin, which is a calpain-mediated breakdown product, increased in relation to the motor dysfunction. There was no significant increase in the 120-kDa fragment, which is a caspase-3-mediated breakdown product.

 


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  		Figure 5. The relationship between neurologic function and the amount of 145- and 150-kDa fragments of {alpha}-fodrin. The levels of {alpha}-fodrin were quantitated by densitometry. Irrespective of the time of evaluation, the rabbits with poor motor function had greater amounts of {alpha}-fodrin fragments (145 and 150 kDa), and the amount of fragments increased according to worsening of the neurologic score.

 
The smearing patterns of DNA breakdown were detected in rabbits with moderate to severe motor dysfunction at 24 and 48 h after reperfusion, but no DNA laddering patterns were detected in any rabbits at any time point (Fig. 6).



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  		Figure 6. Electrophoresis of the genomic DNA in the lumbar spinal cord of the sham control group (C) and three ischemic groups. No laddering pattern was observed in any rabbit. Smearing patterns were detected at 24 and 48 h after reperfusion. Standard lane shows ladder markers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we observed delayed motor dysfunction in approximately two thirds (one fourth for delayed paraplegia) of the rabbits that were subjected to 15-minute spinal cord ischemia produced by a temporal clamping of the abdominal aorta. Neurologic dysfunction tended to develop over 24 to 48 hours after reperfusion, with the observation being consistent with our previous studies where the rabbits were subjected to 20–25 minutes of ischemia (3–5). Histopathological and biochemical examination showed no evidence of apoptosis of the motor neurons but showed characteristics of necrotic neuronal death in our model.

Apoptosis was originally defined as a genetically regulated type of cell death that is fundamental to embryonic development. However, this type of cell death occurs in various pathological states, including postischemic conditions. Its characteristic morphology includes cytoplasmic shrinkage, plasma membrane budding (apoptotic body), nuclear chromatin condensation, DNA fragmentation, and little inflammatory response (10).

In the present study, the morphological changes of the motor neuron at the early stage (eight hours after reperfusion) observed in three of seven rabbits were swelling of the cell body and finely granular dispersed Nissl substance without apparent changes of the nucleus or nucleolus. The destruction of the gray matter observed in paraplegic rabbits at 48 hours after reperfusion was associated with a prominent inflammatory cell infiltration. The nucleolus of the motor neuron was present until marked destruction of the neurons occurred. These changes are the characteristic of necrosis (10). Although, with TUNEL staining, there were a few brownish-stained motor neurons in 3 of 7 rabbits 48 hours after reperfusion, but they showed diffuse brown staining in both the cell nucleus and cytoplasm, which is not an apoptotic change. There were no motor neurons with apoptotic morphological features in any rabbits exhibiting variable degrees of hind limb motor dysfunction.

The amount of 120-kDa fragment generated from {alpha}-fodrin did not increase after ischemia. In contrast, the amount of 145- and 150-kDa fragments increased. The 120-kDa fragment of {alpha}-fodrin is thought to be specific to caspase-3-mediated cleavage and therefore is a specific marker of apoptosis (17). Thus, it is unlikely that the neuronal death in our model was caused by apoptosis. We cannot completely exclude the possibility that non-caspase-3-mediated apoptosis occurred because the 145- and 150-kDa fragments are calpain-mediated and can be produced with either apoptosis or necrosis (17). However, we did not detect a DNA laddering. Taking the morphological and biochemical results together, we suggest that necrotic rather than apoptotic motor neuron death accounts for the delayed onset motor dysfunction after transient spinal cord ischemia.

Our results contradict to those of Sakurai’s group (8,9,11). In that group, they demonstrated delayed onset paraplegia in rabbits in association with apoptotic motor neuron death (TUNEL-stained motor neurons and DNA laddering, both peaking at two days after reperfusion) (8). In separate studies, they demonstrated expression of caspase-3 (9) and Fas antigen (11) at eight hours after ischemia. Although there is no clear explanation for the discrepancy between the results by Sakurai’s group (8,9,11) and ours, the ischemic insult in their studies may have been weaker than ours. Despite the same ischemia duration (15 minutes), hind limb motor function in Sakurai’s study (8) was much better than ours two days after ischemia and further deteriorated seven days after ischemia. In our preliminary study, we observed the rabbits for seven days after reperfusion from 15 minutes of ischemia. However, motor dysfunction was established within 48 hours after reperfusion with no remarkable changes thereafter. The difference in the severity of ischemia may be related to the aortic occlusion method and temperature control. Sakurai’s group (8,9,11) induced ischemia by inflation of a balloon placed in the abdominal aorta, whereas we occluded the abdominal aorta directly by an occluder tube retroperitoneally placed. Sakurai’s group (8,9,11) only monitored the rectal temperature and maintained it at 37°C, whereas we maintained paravertebral muscle temperature at 38°C (esophageal temperature at {approx}38.5°C, which is the normal temperature for rabbits). In addition, the spinal cord at L2 and L3 levels, at which they were examined, might not be the exact central area subjected to severe ischemia. In the study that measured spinal cord blood flow in the abdominal aorta occlusion model in the rabbit, the major flow reduction area was shown to be L5-7 segments of the spinal cord (18). The difference in blood pressure during the periischemic period may be another factor that influenced the severity of ischemic insult. The comparison was not possible because no physiologic data were presented in the Sakurai group reports (8,9,11). Shorter durations of ischemia (i.e., 10 minutes) showed no development of paraplegia in our laboratory (unpublished).

Mackey et al. (7) reported in rabbits that delayed neuronal death after 40 minutes of ischemia was associated with the apoptotic changes (30%–40%) confirmed by histological and biochemical evaluations. They implicated the possibility that apoptosis may help to explain clinical cases of delayed paraplegia. However, their animals showing apoptotic cell death exhibited acute paraplegia not delayed paraplegia. Therefore, it is difficult to accept the concept that apoptosis is the cause of delayed paraplegia.

In our investigation, a few neurons with apoptotic morphological features were recognized in laminae V-VII. The significance of these neurons in laminae V-VII is not known. Neurons in laminae V-VII are interneurons (not motor neurons), and if these neurons are selectively damaged with motor neurons preserved, spastic paraplegia will occur (19). However, in the present study, our rabbits exhibited flaccid paraplegia with characteristic changes of motor neurons compatible with those of necrosis, which probably developed progressively after reperfusion, and the overall time course of these changes correlated well with the progression of motor dysfunction. Therefore, the existence of a few neurons with apoptotic morphological features in laminae V-VII is not likely to be related to the delayed paraplegia.

Jacobs et al. (6) demonstrated that destruction of the blood-spinal cord-barrier and subsequent spinal cord edema, which occurs several hours after reperfusion, may contribute to delayed paraplegia. However, the authors did not discuss what types of cell death contributed to the delayed paraplegia. Our previous study demonstrated significant increases in glutamate concentrations of cerebrospinal fluid microdialysates during spinal cord ischemia (20 minutes of ischemia) and that hypothermia prevented the increase in glutamate concentration and motor dysfunction as well as neuronal death (4). A recent view suggests the possibility that mitochondrial dysfunction may play an important role for delayed neuronal death, but not apoptotic death, after ischemia (20). Delayed increases in intracellular calcium in association with {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor activation may be speculated (21,22). Further studies are required to elucidate the precise mechanism for delayed onset paraplegia after transient spinal cord ischemia.

Although the possibility of apoptotic motor neuron death cannot be completely excluded, apoptosis has a negligible role in the pathophysiology of delayed paraplegia in the model examined.


   Acknowledgments
 
Supported, in part, by a grant-in-aid for scientific research (grant no. 11470323) from the Japanese Ministry of Education, Culture, Sports and Technology.

The authors thank Dr Toshikazu Gondo (First Department of Pathology) for his advice in the assessment of histopathology.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Accepted for publication October 29, 2002.




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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