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Esmolol and Landiolol, Selective ß1-Adrenoreceptor Antagonists, Provide Neuroprotection Against Spinal Cord Ischemia and Reperfusion in Rats

Umehara, Shino MD; Goyagi, Toru MD, PhD; Nishikawa, Toshiaki MD, PhD; Tobe, Yoshitsugu BS; Masaki, Yoko PhD

doi: 10.1213/ANE.0b013e3181cdb06b
Neuroscience in Anesthesiology and Perioperative Medicine: Research Reports
Chinese Language Editions

BACKGROUND: Paraplegia is a devastating and unpredictable complication occasionally resulting from surgery of the thoracic and thoracoabdominal aorta. Because ultrashort-acting selective β1-adrenoreceptor antagonists provide neuroprotective effects after brain ischemia, we hypothesized that they would also ameliorate spinal cord injury after transient ischemia and reperfusion in rats.

METHODS: Male Sprague-Dawley rats were randomly assigned to one of the following 4 groups: saline (received IV infusion of 0.9% saline at a rate of 0.5 mL/h, n = 8), esmolol (esmolol 200 μg/kg/min, n = 8), landiolol (landiolol 50 μg/kg/min), or sham surgical (n = 6). Infusion of saline or drugs was initiated 30 minutes before spinal cord ischemia and continued for the subsequent 24-hour reperfusion. Spinal cord ischemia was induced by intraaortic balloon occlusion combined with proximal arterial hypotension for 10 minutes. The spinal cord was then reperfused for 24 hours. Ischemic injury was assessed in terms of the motor deficit index score of the hindlimb and the number of viable motor nerve cells in the anterior spinal cord at 24 hours after reperfusion.

RESULTS: The motor deficit index scores were significantly lower in the esmolol and landiolol groups compared with the saline group (P < 0.05). Histopathologic evaluation of the spinal cord showed less damage in the esmolol and landiolol groups than in the saline group (P < 0.05).

CONCLUSIONS: These data show that ultrashort-acting selective β1-adrenoreceptor antagonists can reduce neurological injury in a rat model of spinal cord ischemia-reperfusion.

Published ahead of print January 26, 2010 Supplemental Digital Content is available in the text.

From the Department of Anesthesia and Intensive Care Medicine, Akita University Graduate School of Medicine, Akita, Japan.

Presented in part at the 2009 Annual Meeting of the American Society of Anesthesiologists, New Orleans, LA, October 18, 2009.

Disclosure: The authors report no conflict of interest.

Address correspondence and reprint requests to Toru Goyagi, MD, PhD, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. Address e-mail to tgoyagi@doc.med.akita-u.ac.jp.

Accepted November 16, 2009

Published ahead of print January 26, 2010

Paraplegia is a devastating and unpredictable complication of thoracic or thoracoabdominal aortic surgery. According to 2 reviews, the rate of occurrence of paraplegia is 2% to 18%.1,2 Spinal cord ischemia during aortic cross-clamping is inevitable; therefore, a number of intraoperative strategies have been implemented to reduce this complication. They include the use of a left heart bypass, reattachment of segmental intercostals and lumbar arteries, hypothermia, drainage of cerebrospinal fluid, administration of neuroprotective drugs, and monitoring of somatosensory- and motor-evoked potentials.2 These strategies seem to be partly helpful in reducing the risk of spinal cord deficits. However, even after implementing these different approaches, paraplegia remains a persistent complication.

In previous studies, several β-adrenoreceptor antagonists were found to have neuroprotective effects on ischemia and reperfusion injury.37 Landiolol and esmolol, ultrashort-acting selective β1-adrenoreceptor antagonists,8,9 can be used in controllable therapy for the emergency treatment of tachycardia in the perioperative period, because they have a short period of activity and a low cardiodepressant effect. We demonstrated the neuroprotective effect of esmolol and landiolol after transient focal cerebral ischemia in rats.10 However, most studies are performed using the cerebral ischemic model, and there is little understanding regarding the efficacy of these antagonists in spinal cord ischemia and reperfusion.

Based on these considerations, the aim of this study was to evaluate the neuroprotective effect of selective β1-adrenoreceptor antagonists on spinal cord ischemia and reperfusion injury. In this study, we investigated the protective efficacy of esmolol and landiolol in terms of neurological and histopathologic outcomes of spinal cord ischemia and reperfusion in rats.

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METHODS

This study was approved by the Animal Experiment Committee of Akita University Graduate School of Medicine. Male Sprague-Dawley rats weighing 350 to 450 g were housed in polycarbonate cages and had free access to food and tap water. None of the animals had any neurological abnormality before anesthesia and surgery. On the day of surgical preparation, the animals were weighed and then placed in an acrylic plastic box with a continuous flow of 4% halothane and 60% nitrous oxide in oxygen until a deep level of anesthesia was achieved. Anesthesia was maintained with 0.75% to 1.5% halothane in a nitrous oxide/oxygen mixture inhaled with a nonsealing facemask device. The body temperature was continuously monitored with a needle-type thermistor inserted into the paravertebral muscle at the L3-4 levels and was maintained at approximately 38.0°C with a heating lamp and an underbody heating pad. We used the experimental model of spinal cord ischemia and reperfusion described by Taira et al.11 in a previous report. The femoral vein was cannulated with a PE-50 catheter to infuse test drugs or saline, and the catheter was tunneled subcutaneously and exteriorized through a swivel sutured over the dorsal midthorax that allowed the rat to move freely in the cage after emergence from anesthesia. The tail artery was cannulated with a PE-50 catheter for monitoring the distal part of arterial blood pressure (DAP) and for intraarterial infusion of heparin. A PE-60 catheter was inserted into the right carotid artery for monitoring the proximal part of arterial blood pressure (PAP) and was connected to an external blood reservoir positioned at a height of 54 cm above the level of the rat's body to reduce the mean PAP during aortic occlusion to 40 mm Hg. To induce spinal cord ischemia, the right femoral artery was exposed, and a Fogarty 2F balloon-tipped catheter (Edwards Lifesciences, Irvine, CA) was advanced into the thoracic descending aorta (11 cm from the site of insertion). Immediately after performing arterial cannulation, 200 U heparin (0.2 mL) was injected into the tail artery. The catheter balloon was inflated with 0.05 mL saline and maintained for 10 minutes. The efficiency of the occlusion was evidenced by a decrease in the DAP measured at the tail artery. The PAP was controlled at 40 mm Hg during occlusion by drawing blood from the carotid artery into a 20-mL syringe containing 1 mL of 7% sodium bicarbonate solution. After ischemia, the balloon was deflated, and the collected blood was administered to the animals through the carotid artery catheter within 2 minutes.

The rats were randomly divided into 4 groups as follows: 0.9% NaCl solution in the saline group, esmolol at a rate of 200 μg/kg/min in the esmolol group, landiolol at a rate of 50 μg/kg/min in the landiolol group (n = 8 in each group), and sham surgical group (n = 6). The drugs or saline infusion volume was adjusted to a rate of 0.5 mL/h and administered 30 minutes before aortic occlusion until the end of the subsequent 24-hour reperfusion. The rats in the sham surgical group received saline through the catheters inserted in the same manner, but spinal cord ischemia was not induced. Hemodynamic variables (PAP, DAP, and heart rate) were continuously monitored and recorded after surgical preparation for up to 5 minutes after reperfusion. Arterial blood gases and blood glucose were determined just before aortic occlusion and 5 minutes after reperfusion.

After obtaining all the measurements, all catheters except the femoral vein catheter were removed, and the incisions were closed. After anesthesia was discontinued, the animals were allowed to recover and were returned to their cages.

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Neurological Evaluation

At 24 hours after reperfusion, the animals were assessed for their motor deficit index (MDI) with the following grading systems.11 The MDI was quantified by the assessment of ambulation using the hindlimbs and by the placing/stepping reflex. Ambulation using lower extremities was graded as follows: 0, normal (symmetrical and coordinated ambulation); 1, toes flat beneath the body when walking but presence of ataxia; 2, knuckle walking; 3, unable to knuckle walk but some movement of the lower extremities; and 4, no movement of the lower extremities. The placing/stepping reflex was assessed by dragging the dorsum of the hindpaw along the edge of a surface; this evoked a coordinating lifting and placing response (i.e., stepping), which was graded as follows: 0, normal; 1, weak; and 2, no stepping. The MDI scores were calculated for each rat as the sum of the ambulation and placing/stepping reflex scores; the maximal score was 6. The assessments were made by the same observer (YT) who was unaware of the treatment group.

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Histological Evaluation

After scoring neurological function, the animals were anesthetized with 4% halothane in an acrylic plastic box. A high dose of pentobarbital (80 mg/kg) was administered by intraperitoneal injection. After the administration of a direct left ventricular bolus of 0.2 mL heparin, each rat was transcardially perfused with 100 mL heparinized saline followed by 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4). The lumbar spinal cord was removed and postfixed in the same fixative for another 48 hours. After postfixation, the L4 spinal segment was dissected and embedded in paraffin; subsequently, serial transverse sections of 3-μm thickness were prepared. The slides were stained with hematoxylin and eosin and Nissl for quantitative evaluation. Analysis of the degree of ischemic cell injury was based on the number of viable neurons in the ventral part of the gray matter (anterior to a transverse line drawn through the central canal) with ×400 magnification in each group. Cells that contained Nissl substance in the cytoplasm, loose chromatin, and prominent nucleoli were considered to be viable neurons. The number of visible neurons in each animal was taken as the average of 3 sections.

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Statistical Analyses

Physiological variables and the number of normal ventral cells are expressed as the mean ± SD. Comparisons among groups were made with 1-way analysis of variance for multiple comparisons followed by a Bonferroni post hoc test. Comparisons within groups were performed using the paired t test. The data of the MDI scores of animals were expressed as the median with the range in parentheses, and differences were determined by nonparametric analysis using the Kruskal-Wallis test followed by Steel-Dwass post hoc test. A P value <0.05 was considered statistically significant.

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RESULTS

Physiological variables and arterial blood gas data are presented in Table 1. No differences were noted among the experimental groups with respect to the mean PAP, DAP, heart rate, and paravertebral muscle temperature. Heart rate significantly decreased during ischemia in all groups except the sham surgical group. Blood chemistry values before aortic occlusion were within the normal range, and no significant differences were observed within the groups in this regard. Blood glucose and pH levels after reperfusion were significantly higher and lower, respectively, than those before ischemia (P < 0.01).

Table 1

Table 1

Hindlimb function based on the MDI is shown in Figure 1. The medians (range) of MDI scores were significantly higher in the spinal cord ischemia-induced rat groups than the sham surgical group (P < 0.05). The medians and ranges of MDI scores were 3.5 (3–6) and 4.0 (1–6) in the esmolol and landiolol groups, respectively; these values were significantly lower than 6.0 (5–6) in the saline group (P < 0.05).

Figure 1

Figure 1

Representative light photomicrographs of the ventral part of Nissl-stained transverse sections taken from the L4 spinal segment are shown in Figure 2. The number of viable motor nerve cells present in the ventral part of the gray matter at 24 hours after reperfusion is shown in Figure 3. There were more viable motor nerve cells in the esmolol (51 ± 13) and landiolol (39 ± 13) groups than in the saline group (20 ± 14; P < 0.05), whereas there was no significant difference between the esmolol and the sham surgical (64 ± 7) groups.

Figure 2

Figure 2

Figure 3

Figure 3

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DISCUSSION

In this study, we demonstrated a significant improvement in the motor and histopathologic outcomes at 24 hours after ischemia and reperfusion of the spinal cord in rats pretreated with esmolol and landiolol. To our knowledge, this is the first study demonstrating the neuroprotective effect of esmolol and landiolol in spinal cord ischemia and reperfusion.

Neuroprotection by various β-adrenoreceptor antagonists is provided through different mechanisms. Propranolol has beneficial effects in acute focal cerebral ischemia; it improves cerebral oxidative phosphorylation and lipid synthesis in humans and cats.3,4 Propranolol also restores axonal function after a spinal cord injury by suppressing glial scar formation and astrocyte hypertrophy.11 Similarly, carvedilol provides neuroprotection in brain ischemia through its antioxidant and antiapoptotic properties,5,6 whereas nebivolol has a protective effect in spinal cord ischemia via its free radical scavenging and antioxidant activity.7

The cardioprotective effects of esmolol and landiolol during myocardial ischemia and reperfusion have been investigated more precisely in rat and rabbit heart models.1214 It has been postulated that cellular mechanisms underlying esmolol-induced myocardial protection are based on modulation of free radical–mediated reactions and arachidonic acid metabolism, and inhibition of neutrophil superoxide generation and platelet aggregation.1214 However, high-dose landiolol has been demonstrated to ameliorate myocardial dysfunction in isolated and reperfused ischemic heart by restoring adenosine triphosphate degradation.14 Initiation of ischemic arrest is associated with the accelerated release of endogenous catecholamines, which in turn increases neuronal metabolism and aggravates glutamate excitotoxicity. Furthermore, an ischemic cascade can be stimulated in the spinal cord by metabolic bursts, wherein oxygen is reduced to superoxide, hydrogen peroxide, and hydroxyl radicals.15 Sutin and Griffith16 reported that propranolol controlled the activation of astrocytes by antagonism of the β-adrenergic receptor and reduced reactive gliosis in the spinal cord, which accordingly led to restoring axonal function and inhibiting degenerative change after a spinal cord injury. Although the complete mechanism underlying these benefits remains unknown, our finding suggests that the IV administration of esmolol or landiolol had a beneficial effect in the spinal cord as well as in the heart and brain in an animal model.

There are some limitations of this study. First, in our study, we investigated only the short-term effects of esmolol and landiolol in spinal cord ischemia and reperfusion injury. In a clinical setting, paraplegia may develop 1 to 5 days after reperfusion. Spinal cord injury may occur in the acute or delayed phase. Although the exact mechanism is not completely understood, it is believed that the cause of the acute phase is ischemia from hypoperfusion during aortic cross-clamping, and the delayed phase is attributed to embolization or thrombosis of the anterior spinal artery, postoperative hypotension, deleterious effects of leukocytes and microglia, and free radical production.7 We cannot conclude whether esmolol and landiolol bring about any functional improvement as a long-term effect, and further studies are warranted. Second, there is also a possibility that the doses of the β-adrenoreceptor antagonists were not equipotent with the β1-receptor subtype potency. Although it has been reported that landiolol is 6 to 8 times more potent as a β-adrenoreceptor antagonist than esmolol,8 the blood esterase activity is much greater in rats than in dogs and humans.17 Thus far, there is only 1 report on the neuroprotective efficacy of esmolol and landiolol10; accordingly, we selected the same dosage as mentioned in the previous report and did not measure the blood concentration of these drugs. Third, our data showed that in the reperfusion phase, hyperglycemia and acidemia were induced by ischemic stress in the animals. Because there were no significant differences among the experimental groups, it is certain that these differences had little influence on the results of this study. Finally, we did not measure arterial blood pressure and heart rate after 5 minutes of reperfusion; therefore, there is a possibility that hemodynamic change had occurred after animals recovered in their cages, which might affect the results. However, the administration of esmolol or landiolol attenuated the spinal cord damage after ischemia and reperfusion.

In conclusion, this study demonstrated that pretreatment with the ultrashort-acting selective β1-adrenoreceptor antagonists, esmolol and landiolol, attenuated the damage caused by spinal cord ischemia in rats. Motor function was better and there were few histopathologic changes in the rats receiving esmolol or landiolol compared with the control saline group. Because we investigated the short-term effect of esmolol and landiolol during the acute phase in our current study, we cannot conclude whether these β1-adrenoreceptor antagonists produce any functional improvements as a long-term effect. Therefore, further study should be performed to evaluate the long-term as well as short-term neuroprotective effects of esmolol and landiolol. The underlying mechanisms and the clinical implications of these beneficial effects need to be further investigated.

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STUDY FUNDING

Supported by Institutional funds from the Department of Anesthesia and Intensive Care Medicine, Akita University Graduate School of Medicine.

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ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical advice of Dr. Manabu Kakinohana (Department of Anesthesiology, University of Ryukyu, Japan).

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