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Reduction of Spinal Cord Ischemia/Reperfusion Injury with Simvastatin in Rats

Saito, Takeshi, PhD*; Tsuchida, Masanori, MD, PhD*; Umehara, Shino, MD; Kohno, Tatsuro, MD, PhD; Yamamoto, Hiroshi, MD, PhD§; Hayashi, Jun-ichi, MD, PhD*

doi: 10.1213/ANE.0b013e318224ac35
Neuroscience in Anesthesiology and Perioperative Medicine: Research Reports
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SDC

BACKGROUND: Surgery of the thoracic or thoracoabdominal aorta may cause spinal cord ischemia and subsequent paraplegia. However, conventional strategies for preventing paraplegia due to spinal cord ischemia provide insufficient protection and cause additional side effects. We hypothesized that simvastatin, a drug recently shown to be neuroprotective against brain ischemia/reperfusion, would be neuroprotective in a rat spinal cord ischemia/reperfusion model.

METHODS: Rats were randomly assigned to simvastatin, vehicle, or sham-surgery (sham) groups (n = 6 per group). Simvastatin (10 mg/kg) or vehicle was administered subcutaneously once daily for 7 days before aortic balloon occlusion, and once at 24 hours after reperfusion. Spinal cord ischemia was induced by balloon inflation of a 2F Fogarty catheter in the thoracic aorta, and the proximal mean arterial blood pressure was maintained at 40 mm Hg for 12 minutes. The sham group received the same operation without inflation of the balloon. Ischemic injury was assessed by hindlimb motor function using the Motor Deficit Index score at 6 to 48 hours after ischemic reperfusion, and histological assessment of the spinal cord was performed 48 hours after reperfusion.

RESULTS: The Motor Deficit Index scores at 24 and 48 hours after reperfusion were significantly improved in the simvastatin group compared with the vehicle group (P = 0.021 and P = 0.023, respectively). Furthermore, there were significantly more normal motor neurons in the simvastatin group than in the vehicle group (P = 0.037). The percentage area of white matter vacuolation was significantly smaller in the simvastatin group than in the vehicle group (P = 0.030).

CONCLUSIONS: Simvastatin treatment can attenuate hindlimb motor dysfunction and histopathological changes in spinal cord ischemia/reperfusion injury in rats.

Published ahead of print June 16, 2011

From the Divisions of *Thoracic and Cardiovascular Surgery, and Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata; and Departments of Anesthesia and Intensive Care Medicine, and §Cardiovascular Surgery, Akita University School of Medicine, Akita, Japan.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Takeshi Saito, PhD, Division of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. Address e-mail to saito@med.niigata-u.ac.jp.

Accepted May 11, 2011

Published ahead of print June 16, 2011

Surgery of the thoracic or thoracoabdominal aorta may cause spinal cord ischemia during aortic cross-clamp and subsequent paraplegia, a serious postoperative complication.1,2 Conventional strategies for preventing paraplegia due to spinal cord ischemia, including systemic hypothermia, left heart bypass, spinal fluid drainage, and ischemic preconditioning, do not provide sufficient protection, and may cause additional unfavorable complications because of their complexity and invasiveness.3,4

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are frequently used for reducing serum cholesterol. Several large-scale clinical studies reported that statins also reduced the frequency of cerebral infarction.5,6 The mechanism of action was initially considered to be via regulation of the tone and plaque levels of vessels by reducing cholesterol, a risk factor for stroke, to normal or lower average levels.5,6 However, a cerebroprotective action of statins that is not fully explained by decreased blood cholesterol was also reported in several studies.7,8 Furthermore, statins can induce ischemic tolerance to neurons both in vitro and in vivo.715 However, there are no studies assessing the neuroprotective actions of statins on spinal cord ischemia.

Simvastatin has a well-established safety profile for patients with hyperlipidemia. Experimental studies also suggest that simvastatin exhibits greater neuronal protective effects than other statins.7,9 For example, Zacco et al.9 demonstrated that simvastatin reduced N-methyl-D-aspartate–induced excitotoxicity in cortical neurons more than atorvastatin, mevastatin, or pravastatin. In addition, simvastatin is liposoluble and crosses the blood-brain barrier easier than other water-soluble statins.16 Therefore, we investigated the effect of simvastatin treatment on neurological and histopathological outcomes in a spinal cord ischemia/reperfusion model in rats.

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METHODS

Animal and Study Groups

This study was approved by the Animal Experiment Committee of Niigata University Graduate School of Medical and Dental Sciences. Male Sprague-Dawley rats weighing 370 to 440 g were housed and maintained on a 12-hour light/dark cycle with free access to food and water. Rats were randomly divided into the simvastatin (n = 6), vehicle (n = 6), and sham surgery (sham, n = 6) groups. In the simvastatin group, simvastatin (10 mg/kg) was administered subcutaneously once daily for 7 days before occlusion, and once at 24 hours after reperfusion. In the vehicle and sham groups, the same volume of vehicle was administered subcutaneously using the same timing regime as for the simvastatin group. All groups received the same surgery, but spinal cord ischemia was not induced in the sham group. Because simvastatin is converted to an active form by inactive lactone by alkaline hydrolysis in the blood, we pretreated simvastatin by alkaline hydrolysis12 before administration.

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Simvastatin Pretreatment

Simvastatin (4.0 mg; Wako Chemicals, Osaka, Japan) was dissolved in 0.1 mL of 95% ethanol. Next, 0.15 mL of 0.1 N NaOH was added and the solution was heated at 50°C for 2 hours. The solution was then neutralized with HCl to pH 7.2 to 7.3. The final volume was made up to 1 mL with distilled water.12

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Surgery

Spinal cord ischemia/reperfusion was induced as reported by Taira and Marsala.17 Rats were anesthetized in an acrylic box with 4% isoflurane in an air/oxygen mixture. After induction, the rats were maintained with mixed gas (1 L/min air, 1 L/min oxygen, and 1.5%–2% isoflurane) delivered by an inhalation mask. A PE-50 cannula was inserted into the tail artery for monitoring distal arterial blood pressure. To control the proximal arterial blood pressure at approximately 40 mm Hg during the period of aortic occlusion, a PE-60 cannula connected to an external blood reservoir (38.0°C) positioned at 54 cm above the rat's body was inserted into the left carotid artery to reduce arterial blood pressure. The left femoral artery was isolated and a 2F Fogarty catheter was placed into the descending thoracic aorta so that the tip reached directly below the left subclavian artery (11 cm distal from the site of insertion). A thermocouple probe was placed in the paravertebral muscle at the level of T10-11 to monitor the paravertebral muscle temperature. The paravertebral muscle temperature during aortic occlusion was maintained between 37.0°C and 37.2°C using an underbody heating pad and a heat lamp. At the completion of all cannulation, heparin (200 IU) was injected into the tail artery. To induce spinal cord ischemia, the balloon catheter was inflated with 0.05 mL saline, and blood was allowed to flow into the external reservoir. The efficiency of the occlusion was established by an immediate and sustained loss of detectable pulse and a decrease of distal blood pressure. After 12 minutes of spinal cord ischemia, the balloon was deflated and the blood was reinfused over a 120-second period. Protamine sulfate (4 mg) was then administered subcutaneously. Blood gases, pH, glucose, and hemoglobin were measured at 1 minute before and 10 minutes after the ischemia using a blood gas analyzer (Cobas b 221; Diagnostik, Tokyo, Japan). All arterial lines were then removed and incisions were closed. After anesthesia was discontinued, the animals were allowed to recover and returned to their cages.

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Neurological Evaluation

Hindlimb motor function was assessed at 6, 12, 24, and 48 hours after reperfusion using the Motor Deficit Index (MDI) score,17 which was quantified by ambulation and placing/stepping reflex by investigators who were blinded to group information. Ambulation using the hindlimbs was graded as follows: 0 = normal (symmetrical and coordinated ambulation); 1 = toes flat under body when walking but ataxia present; 2 = knuckle walking; 3 = unable to knuckle walk but some movement of the hindlimbs; and 4 = no movement or drags lower extremities. The placing/ stepping reflex was assessed based on the dragging movements and responses of the hindpaw dorsum when touching the floor surface. A coordinating lifting and placing response (i.e., stepping), which was generally evoked when a hindpaw touched the ground, was graded as follows: 0 = normal; 1 = weak; and 2 = no stepping. MDI score was calculated for each rat as the sum of both the above scores at each time interval. The maximal MDI score was 6 (score of 4 for ambulation and 2 for the placing/stepping reflex).

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Histopathology

After evaluation of motor behavior, animals were anesthetized with isoflurane, and 100 mg/kg pentobarbital was administered intraperitoneally. Animals were then transcardially perfused with 100 mL heparinized saline, followed by 150 mL of 10% phosphate-buffered formalin. The lumbar spinal cord was removed and fixed with the same fixative for another 2 to 7 days. After fixation, the fourth lumbar spinal segment was dissected and embedded in paraffin, and 3-μm-thick serial transverse sections were prepared. The slides were stained with hematoxylin and eosin (HE) and Nissl for quantitative evaluation. For analysis, 3 representative sections were taken from segments of the fourth lumbar cord with 100-μm interspaces.

Gray matter damage was assessed on the basis of the number of normal motor neurons in the ventral part of the gray matter (anterior to a transverse line drawn through the central canal) at ×400 magnification. Cells that contained Nissl substance in the cytoplasm, loose chromatin, and prominent nucleoli were considered normal motor neurons. The number of normal motor neurons in each animal was obtained by averaging counts from the 3 different slides dyed with Nissl. White matter damage was assessed based on the extent of vacuolation in the ventral and ventrolateral white matter that was stained with HE. A 0.04 mm2 area in the ventral and ventrolateral white matter was used for assessment as reported by Kurita et al.18 Each target area in the white matter was divided into 64 subareas. The number of subareas that were >75% occupied by vacuoles was counted, and the percentage area of vacuolation was calculated (×400). The percentage area of vacuolation in each animal was obtained by averaging 2 areas of the right and left hemicords of the 3 different slides (a total of 12 areas). The number of normal neurons and the percentage area of vacuolation were counted by an observer unaware of group assignment.

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Statistical Analysis

Statistical analyses of physiological data, the number of normal neurons, and the percentage area of vacuolation in the spinal cord were performed using Tukey's method for all possible pairwise comparisons. Neurological outcomes among experimental groups at individual time points after reperfusion were assessed using an exact nonparametric test (Kruskal-Wallis test) followed by the Steel-Dwass post hoc test. The relationships among neurological outcome, the number of normal neurons, and the percentage area of vacuolation were analyzed by Spearman rank correlation coefficient. A P value of <0.05 was considered significant.

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RESULTS

Physiological data are shown in Table 1. There were no differences in pH, PaO2, PaCO2, hemoglobin, glucose, or mean distal arterial pressure among the groups before ischemia. During ischemia, mean distal arterial pressure values in the simvastatin and vehicle groups were significantly lower than those in the sham group (P < 0.001). After reperfusion, the pH value in the sham group (7.33 ± 0.08) was significantly higher than in the simvastatin (7.19 ± 0.09) and vehicle (7. 21 ± 0.09) groups (P = 0.006 and P = 0.015, respectively). During and after the ischemic period, there were no differences in paravertebral muscular temperature among the groups. Hindlimb function based on MDI is shown in Figure 1. MDI scores at 24 and 48 hours were significantly different (Kruskal-Wallis: both P < 0.001) among groups. MDI scores were significantly lower in the simvastatin group than in the vehicle group at 24 and 48 hours after ischemia (Steel-Dwass: P = 0.021 and P = 0.023, respectively).

Table 1

Table 1

Figure 1

Figure 1

Representative light photomicrographs of the ventral gray matter of Nissl-stained transverse taken from the L4 spinal cord segment are shown in Figure 2A. There were more normal motor neurons at 48 hours after reperfusion in the simvastatin group (43 ± 14) than in the vehicle group (19 ± 18; P = 0.037) (Fig. 2B). There were significantly more normal motor neurons in the sham group (72 ± 12) than in the simvastatin group (P = 0.010).

Figure 2

Figure 2

Representative light photomicrographs of HE-stained sections in the ventrolateral white matter are shown in Figure 3A. Vacuolation in the white matter was widespread and prominent in the vehicle group, whereas only small and scattered vacuolation was observed in the simvastatin group. The percentage area of vacuolation in the simvastatin group (10% ± 4%) was significantly smaller than in the vehicle group (19% ± 5%; P = 0.030) (Fig. 3B). The sham group (1% ± 1%) had a significantly smaller percentage area of vacuolation than the simvastatin group (P = 0.003).

Figure 3

Figure 3

The relationships among MDI scores, the number of normal neurons, and percentage areas of vacuolation are shown in Figure 4. There was a significant negative correlation between MDI scores and the number of normal neurons (with sham group: Spearman correlation coefficient −0.89, P < 0.001; without sham group: Spearman correlation coefficient −0.73, P = 0.008). There was a significant positive correlation between MDI scores and the percentage areas of vacuolation (with sham group: Spearman correlation coefficient 0.84, P < 0.001; without sham group: Spearman correlation coefficient 0.60, P = 0.043). There was a significant negative correlation between the number of normal neurons and the percentage areas of vacuolation (with sham group: Spearman correlation coefficient −0.93, P < 0.001; without sham group: Spearman correlation coefficient −0.91, P < 0.001).

Figure 4

Figure 4

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DISCUSSION

In this study, we demonstrated that simvastatin treatment significantly attenuated hindlimb motor dysfunction and histological damage in the gray and white matter of the spinal cord induced by ischemia/reperfusion in rats. The importance of evaluating both white matter and gray matter injury in spinal cord ischemia has been reported.1820 Follis et al.19 also demonstrated that gray and white matter of the spinal cord exhibit different sensitivities to ischemic injury because of tissue-structural differences.19 Moreover, Kanellopoulos et al.20 reported that (2,3-dihydroxy-)6-nitro-7-sulfamoyl-(f)-quinoxaline-2,3-dione (NBQX), an AMPA/kainate glutamate receptor antagonist, preserved hindlimb motor function in a rat spinal cord ischemia/reperfusion model20; white matter damage was decreased in the NBQX group at 48 hours after reperfusion, whereas there was no improvement in gray matter damage.

In the central nervous system, white matter tissue is highly susceptible to ischemic damage. The myelin sheath accounts for 50% of white matter volume and is susceptible to severe damage by lipid peroxidation because it is composed of 70% to 80% lipid.21 Furthermore, oligodendrocytes are vulnerable to superoxide produced during ischemia/reperfusion because they contain only low levels of superoxide antagonists such as Mu-superoxide and catalase.22 Because simvastatin can inhibit lipid peroxidation and superoxide production after cerebral ischemia/reperfusion,7 the preservation of hindlimb motor function observed in the present study may have been associated with a protective effect of simvastatin against both white and gray matter injury. Although the complete mechanism by which simvastatin protects the spinal cord against ischemia/reperfusion injury is unknown, several mechanisms are suggested for neurons.710,12,13,15 Simvastatin reduces oxygen and glucose deprivation/reoxygenation- or N-methyl-D-aspartate-induced neuronal injury in vitro,9,15 and in rat cerebral ischemia models, cerebral infarction volume and production of 4-hydroxynonenal–conjugated protein in the cerebral cortex and caudate are reduced with simvastatin administration.7 In transient cerebral ischemia rat models, mRNA expression of interleukin-1β and tumor necrosis factor-α are suppressed in the cortex and hippocampus after simvastatin, with improvements in T-maze and circular water maze memory tests.10 These data also suggest that simvastatin should protect spinal cord neurons from ischemia/reperfusion injury. However, this requires further investigation.

There are several reports on the effects of varying timing of statin administration on ischemic neuronal injury.715 For example, simvastatin administration from 1 week to 48 hours before ischemia induces ischemic tolerance in rat cerebral ischemia models.8,10 However, evidence from animal and clinical studies suggests that spinal cord neuronal injury may continuously worsen even after ischemia/reperfusion,2325 and in the present study, the spinal cord continuously deteriorated and near-complete paraplegia developed at 48 hours after reperfusion in the vehicle group. A potential cause of delayed neuronal damage is an overinflammatory response that continues after reperfusion.23 Furthermore, administration of pravastatin after reperfusion was reported to reduce the overinflammatory reaction and was neuroprotective at 5 days' recovery from transient cerebral ischemia in rats.11 Based on these data, we administered simvastatin from 1 week before ischemia and continued for the first 24 hours of postischemic reperfusion. It is possible that a selection of different time points for simvastatin administration may have resulted in different outcomes. Further studies are required to determine the optimal window for treatment.

The concentration of simvastatin used in our study was based on that previously reported,13 although it was higher than that used clinically. However, statin sensitivity differs among species, and the rat is less sensitive to statin than the human.7,8 As such, it may be unnecessary to administer higher doses of statin to induce ischemic tolerance in humans. Rhabdomyolysis is a serious side effect of high-dose statin. However, in the present study, we did not observe abnormal findings in urine that were suggestive of rhabdomyolysis in simvastatin-treated rats. Westwood et al.26 also reported that creatine kinase and muscle necrosis were not observed histologically in rats administered 60 mg/kg/d simvastatin for 43 days.26

A potential limitation of our study is that we only examined the efficacy of simvastatin up to 48 hours after reperfusion because the most severe spinal cord injury occurs at this time.24 In the clinical setting, however, paraplegia may develop at 1 to 5 days after spinal cord ischemia.27 In addition, some patients with cerebral infarction who discontinued chronic statin therapy in its acute phase had a higher mortality rate28,29 and infarct volume28 at long-term follow-up, suggesting that withdrawal from statin administration influences long-term prognosis. However, prophylactic treatment with statins in various animal models of transient cerebral ischemia was reported to reduce postischemic delayed neuronal death in the hippocampus at 3 and 5 days' recovery.3032 Furthermore, a study of permanent cerebral ischemia reported that the rats treated with atorvastatin had reduced infarction volumes at 21 days after ischemia.33 Further studies are required to determine these long-term results including the influence of discontinuation of simvastatin administration.

In summary, simvastatin treatment attenuated hindlimb motor dysfunction at 24 and 48 hours after reperfusion and reduced both white and gray matter injury at 48 hours after reperfusion in our rat spinal cord ischemia model. These data suggest a potential application for simvastatin for neuroprotection in the perioperative management of descending and thoracoabdominal aortic surgery. Further studies on the timing of administration and long-term effects are required.

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DISCLOSURES

Name: Takeshi Saito, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Takeshi Saito has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Masanori Tsuchida, MD, PhD.

Contribution: Data analysis and data collection.

Attestation: Masanori Tsuchida has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Shino Umehara, MD.

Contribution: Data analysis and data collection.

Attestation: Shino Umehara has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Tatsuro Kohno, MD, PhD.

Contribution: Data analysis.

Attestation: Tatsuro Kohno has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Hiroshi Yamamoto, MD, PhD.

Contribution: Data analysis and data collection.

Attestation: Hiroshi Yamamoto has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Jun-ichi Hayashi, MD, PhD.

Contribution: Conduct of study and data collection.

Attestation: Jun-ichi Hayashi has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

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ACKNOWLEDGMENTS

We are grateful to Prof. Shin-ichi Toyabe (Niigata University Crisis Management Office, Niigata University Medical and Dental Hospital) for his valuable help.

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