- Email: [email protected]
Effect of Pretreatment With Simvastatin on Spinal Cord Ischemia-Reperfusion Injury in Rats
Effect of Pretreatment With Simvastatin on Spinal Cord Ischemia-Reperfusion Injury in Rats Jinyoung Hwang, MD,* Jong In Han, MD, PhD,† and Sunghee Han, MD, PhD* Objective: The aim of this study was to evaluate the pretreatment effect of simvastatin on spinal cord ischemiareperfusion injury. Design: Prospective, interventional study. Setting: University research laboratory. Participants: Forty-five male Sprague-Dawley rats. Interventions: Rats were treated with oral simvastatin, 10 mg/kg (simvastatin group; n ⴝ 15) or saline (control group; n ⴝ 15) for 5 days before ischemia. Spinal cord ischemia was induced using a balloon-tipped catheter placed in the proximal descending aorta in the control and simvastatin groups, but not in the sham group (n ⴝ 15). Measurements and Main Results: Neurologic function was assessed daily using the motor deficit index until 7 days after reperfusion. After the last neurologic evaluation, a histologic examination of the spinal cord was performed. At day 1 after reperfusion, the simvastatin group showed a
significantly lower motor deficit index compared with the control group (2.0, 2.0-2.0, v 4.0, 3.5-5.0; p < 0.001). This trend was sustained at day 7 (2.0, 1.5-2.0, v 4.0, 3.0-4.0; p < 0.001). The simvastatin group displayed a significantly larger number of normal motor neurons compared with the control group (mean ⴞ SD, 31.7 ⴞ 6.1 v 20.4 ⴞ 4.4; p < 0.001). However, compared with the sham group, the simvastatin group displayed fewer intact motor neurons (sham group, 38.5 ⴞ 5.1; p ⴝ 0.005). Conclusions: Pretreatment with simvastatin, 10 mg/kg, given orally for 5 days before the ischemia-reperfusion insult, improved the neurologic outcome and preserved more normal motor neurons compared with the control group in a rat model of spinal cord ischemia-reperfusion. © 2012 Elsevier Inc. All rights reserved.
S
10 mg/kg (Zocor, Merck, Whitehouse Station, NJ) mixed with normal saline (1 mL) daily for 5 days before ischemia. Oral administration was performed by oral gavage using a 16-gauge feeding needle. Male Sprague-Dawley rats (300-350 g; n ⫽ 45) were anesthetized in an acryl box with isoflurane (5 vol%) in 100% oxygen. After induction, anesthesia was maintained using a facial mask for the inhalation of 1.0 to 2.5 vol% isoflurane driven by an oxygen flow of 2 L/min. Rats were placed in the supine position and the hair in the left inguinal area and neck was shaved. The tail artery was exposed and cannulated with a polyethylene catheter for heparin injection and distal arterial pressure monitoring. For the induction of spinal cord ischemia, the left femoral artery was exposed, and a 2Fr Fogarty catheter (Fogarty Arterial Embolectomy Catheter, Edwards Lifesciences, Irvine, CA) was inserted into the descending thoracic aorta so the tip of the catheter reached the left subclavian artery (11 cm from the site of insertion). The left carotid artery was exposed and cannulated with a 20-gauge catheter (BD Insyte, Becton Dickinson, Sandy, UT) to monitor the proximal arterial pressure. The catheter was connected to a saline-filled external blood reservoir to control the proximal arterial pressure to 80 mmHg during the aortic occlusion. The body temperature was monitored with a rectal probe inserted 6 cm into the rectum and maintained at 37.5 ⫾ 0.5°C with a heating blanket and overhead lamp. Spinal cord ischemia was induced by investigators who were blinded to the group assignment, using the method of Taira and Marsala20 (Fig 1). After completion of the cannulation, heparin (150 U) was injected into the tail artery and the balloon of the Fogarty catheter was inflated with saline, 0.05 mL. Simultaneously, the blood was drained from the carotid artery
PINAL CORD ISCHEMIA remains a devastating neurologic complication of thoracoabdominal aortic surgery. The incidence of paraparesis or paraplegia after thoracoabdominal aortic aneurysm repair has been reported to be 16%.1 The mechanism of paraplegia after thoracoabdominal aortic surgery has been considered an ischemia-reperfusion injury secondary to aortic clamping and declamping during the surgery. In ischemia-reperfusion injury of the spinal cord, the glutamate-mediated excitotoxicity,2 reactive oxygen species production, inflammation, and apoptosis3 play a key role in neuronal cell death. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), currently used as cholesterol-lowering agents, provide benefits other than lowering lipids, such as anti-inflammatory effects,4 antioxidants,5 and improved endothelial function.6 The beneficial effects of statins on ischemia-reperfusion injury have been reported in various organs, including the brain,7-9 heart,4,10 lungs,11 liver,12 kidneys13 and intestines.14 The effect of statins in mechanical spinal cord injury was reported first by Pannu et al15 who found that the administration of statin resulted in significant decreases of inflammatory cytokines and neuronal cell death and a significant improvement in locomotor scores. However, the potential for statins to promote neurologic recovery after spinal cord mechanical injury has been studied in only a limited number of reports,16-19 and the neurologic benefits are controversial. Furthermore, the pretreatment effect of simvastatin has not been determined clearly. This study was designed to evaluate the pretreatment effect of simvastatin on spinal cord ischemia-reperfusion injury. METHODS All animal experiments and animal care were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The following experimental protocol was approved by the institutional animal care and use committee of a university hospital. Rats were assigned randomly to 1 of 3 groups: (1) the sham group (n ⫽ 15) was administered oral saline (1 mL) daily for 5 days; (2) the control group (n ⫽ 15) was administered oral saline (1 mL) daily for 5 days; and (3) the simvastatin group (n ⫽ 15) received oral simvastatin
KEY WORDS: simvastatin, spinal cord ischemia, thoracoabdominal aortic surgery, paraplegia
From the ⴱDepartment of Anesthesiology and Pain Medicine, Seoul National University, Bundang Hospital, Seongnamsi, Gyeonggido, Korea; and †Department of Anesthesiology and Pain Medicine, School of Medicine, Ewha Womans University, Seoul, Korea. This study was supported by grant number 11-2010-018 from Seoul National University Bundang Hospital research fund. Address reprint requests to Jong In Han, MD, PhD, Department of Anesthesiology and Pain Medicine, School of Medicine, Ewha Womans University, 911, Mok-dong, Yangcheon-gu, Seoul, 158-710, Korea. E-mail: [email protected] © 2012 Elsevier Inc. All rights reserved. 1053-0770/2701-0001$36.00/0 doi:10.1053/j.jvca.2012.01.025
Journal of Cardiothoracic and Vascular Anesthesia, Vol 27, No 1 (February), 2013: pp 79-85
79
80
HWANG, HAN, AND HAN
stepping reflex), and the maximum deficit was indicated by a score of 6. The MDI was evaluated by an observer who was blinded to the group assignment. After the last neurologic assessment, the rats were anesthetized deeply with isoflurane and were perfused transcardially with heparinized saline, 100 mL. In brief, the heart was exposed, a 23-gauge needle was inserted into the left ventricle, and the right atrium was cut. Heparinized saline (100 mL) passed through the needle, circulated around the body, and exited through the open right atrium. The spinal cord was removed and fixed in 10% buffered formalin for 24 hours. Spinal cord segments at the L3-L5 level were embedded in paraffin. Transverse sections were cut at a thickness of 4 m and stained with hematoxylin and eosin. Neuronal injury was evaluated at ⫻200 magnification by an investigator blinded to the group assignment. In paraplegic animals, the anterior spinal cord was destroyed markedly and the disappearance of normal motor neurons was significant. To assess the degree of ischemic neuronal injury, the number of normal motor neurons in the anterior horn of the spinal cord (anterior to a line drawn through the central canal perpendicular to the vertebral axis) was counted in 3 sections for each animal and then averaged.21 SPSS 15 (SPSS Inc, Chicago, IL) was used for statistical analysis. Data were expressed as mean ⫾ SD or median (interquartile range). Physiologic data and the number of motor neurons were compared using a one-way analysis of variance followed by the Dunnett post hoc test. At each time point, the hind limb motor function of the 3 groups was compared with a Kruskal-Wallis test followed by a Mann-Whitney U test. The change in the MDI within each group was compared by the Friedman test followed by a Wilcoxon rank-sum test. Statistical significance was accepted when the p value was ⬍0.05. RESULTS
Fig 1. A diagram illustrating the preparation for spinal cord ischemia in a rat.
into the external reservoir to control the proximal arterial pressure at 80 mmHg by withdrawing blood during the aortic occlusion. The success of the aortic occlusion was confirmed by an immediate decrease and sustained loss of distal arterial pressure. The sham group was prepared in the same manner, but spinal cord ischemia was not induced. After 10 minutes 30 seconds of aortic occlusion, the Fogarty balloon was deflated, and the drained blood was reinfused. After the reperfusion, all catheters were removed and the wounds were closed. The rats were allowed to recover from anesthesia and were returned to their cages. Mean arterial pressure, heart rate, and body temperature were monitored continuously. Values were recorded before the aortic occlusion, 5 minutes after the aortic occlusion, and 10 minutes after reperfusion. Arterial blood gas and hematocrit levels were measured before the aortic occlusion and 10 minutes after reperfusion. Neurologic function was assessed daily until 7 days after reperfusion. The motor function of the hind limb was evaluated by an assessment of the ambulation and placing/stepping reflex according to the method of Taira and Marsala20 (Table 1). The placing/stepping reflex was assessed by dragging the dorsum of the hind paw over the edge of a surface. Normally, this evokes a coordinating lifting and placing/ stepping response. The motor deficit index (MDI) was defined as the sum of the scores (ambulation with lower extremities plus placing/
Mean distal arterial pressure, heart rate, and rectal temperature were similar among the 3 groups at each time point (Table 2). The arterial pH and partial pressures of carbon dioxide and oxygen were not different among the groups at any time point. There was no difference in hematocrit values among the groups before the aortic occlusion. After reperfusion, the control and simvastatin groups showed significantly decreased values of hematocrit compared with the sham group (p ⫽ 0.003, each group v sham group), but there was no difference between the control and simvastatin groups (Table 3). All animals survived until the final neurologic assessment at 7 days after reperfusion. The sham group showed an MDI of 0 throughout the experimental period. On day 1 after reperfusion, the simvastatin group showed a significantly lower MDI com-
Table 1. Assessment of Ambulation and Placing/Stepping Reflex Ambulation (Walking With Lower Extremities)
0 ⫽ normal (symmetric and coordinated ambulation) 1 ⫽ toes flat under body when walking but ataxia present 2 ⫽ knuckle walking 3 ⫽ movement in lower extremities but unable to knuckle walk 4 ⫽ no movement, drags lower extremities
Placing/Stepping Reflex
0 ⫽ normal
1 ⫽ weak 2 ⫽ no stepping
SIMVASTATIN AND SPINAL CORD INJURY
81
Table 2. Hemodynamic Variables and Temperature
Before aortic occlusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15) During aortic occlusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15) After reperfusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15)
MDAP (mm Hg)
HR (beats/min)
Temperature (°C)
99.1 ⫾ 7.6 100.1 ⫾ 11.1 100.0 ⫾ 8.5
313.5 ⫾ 17.7 318.5 ⫾ 22.0 303.3 ⫾ 24.8
37.4 ⫾ 0.2 37.3 ⫾ 0.3 37.5 ⫾ 0.3
No occlusion 6.2 ⫾ 1.3 6.0 ⫾ 1.1
No occlusion 301.1 ⫾ 15.3 290.1 ⫾ 29.7
No occlusion 37.4 ⫾ 0.3 37.6 ⫾ 0.3
103.2 ⫾ 7.4 99.1 ⫾ 14.9 102.5 ⫾ 13.3
315.4 ⫾ 14.8 320.7 ⫾ 21.5 296.8 ⫾ 28.4
37.4 ⫾ 0.2 37.5 ⫾ 0.3 37.5 ⫾ 0.3
NOTE. Values are presented as mean ⫾ SD. The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; the simvastatin group received simvastatin orally for 5 days before the surgery. Aortic occlusion was performed in the control and simvastatin groups. The groups did not show any difference in the measured hemodynamic variables at each time point. Abbreviations: HR, heart rate; MDAP, mean distal arterial pressure.
pared with the control group. This trend was sustained through day 7. Within each group, there was no statistically significant change in the MDI during the study period (Table 4). The number of normal motor neurons in the anterior spinal cord is presented in Fig 2. The simvastatin group displayed a significantly larger number of normal motor neurons compared with the control group (31.7 ⫾ 6.1 v 20.4 ⫾ 4.4, p ⬍ 0.001). However, compared with the sham group, the simvastatin group displayed fewer intact motor neurons (sham group, 38.5 ⫾ 5.1; p ⫽ 0.005). Representative photographs from each group are shown in Fig 3. DISCUSSION
The present study showed that pretreatment with simvastatin 10 mg/kg given orally for 5 days before an ischemia-reperfusion insult improved the neurologic outcome and preserved more normal motor neurons compared with the control group in a rat model of spinal cord ischemia-reperfusion. Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been shown convincingly to produce effects
that are dependent on and independent of lipid lowering.22 They inhibit the conversion of acetyl and acetoacetyl coenzyme A to mevalonate in the formation of cholesterol and prevent the formation of isoprenoids, which are critical proteins in the inflammatory cascade.23 The lipid-lowering independent effects of statins include some beneficial effects on endothelial function and anti-inflammatory actions. Statins increase endothelial nitric oxide production and improve endothelial function by directly upregulating nitric oxide synthase expression24 and decreasing oxidative stress.25 Furthermore, they attenuate leukocyte-endothelial cell interactions26,27 and inhibit the expression of proinflammatory cytokines, including tumor necrosis factor-␣, interleukin-1, and interleukin-6.28 The protective effects of statins have been reported in experimental heart,4,10,29 intestine,14 liver12 and kidney13 ischemia-reperfusion models. In particular, the protective effects of statins have been demonstrated well in the heart. The use of statin therapy for acute myocardial ischemia was related to a significantly lower rate of early complications and in-hospital mortality in human clinical studies.30-32
Table 3. Hematologic Parameters
Before aortic occlusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15) After reperfusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15)
pH
PaO2 (mmHg)
PaCO2 (mmHg)
Hematocrit (%)
7.40 ⫾ 0.02 7.40 ⫾ 0.02 7.39 ⫾ 0.02
259.1 ⫾ 47.5 252.4 ⫾ 39.2 222.0 ⫾ 31.1
35.6 ⫾ 2.2 36.1 ⫾ 3.1 35.4 ⫾ 2.3
39.1 ⫾ 2.9 39.3 ⫾ 2.9 39.4 ⫾ 2.4
7.38 ⫾ 0.02 7.35 ⫾ 0.06 7.35 ⫾ 0.04
269.8 ⫾ 34.4 263.3 ⫾ 29.6 231.7 ⫾ 36.5
35.1 ⫾ 2.4 35.9 ⫾ 3.4 36.1 ⫾ 3.2
39.3 ⫾ 2.2 36.2 ⫾ 2.1* 36.8 ⫾ 2.2*
NOTE. Values are presented as mean ⫾ SD. The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; the simvastatin group received simvastatin orally for 5 days before the surgery. Aortic occlusion was performed in the control and simvastatin groups. The groups did not show any difference in blood gas data at each time point. Hematocrit did not differ among the 3 groups before aortic occlusion; after reperfusion, the control and simvastatin groups showed significantly lower hematocrit values compared with the sham group. Abbreviations: PaCO2, partial arterial pressure of carbon dioxide; PaO2, partial arterial pressure of oxygen. *p ⬍ 0.05 compared with the sham group.
82
HWANG, HAN, AND HAN
Table 4. Motor Deficit Index POD 1
POD 3
POD 7
Sham (n ⫽ 15) 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0) Control (n ⫽ 15) 4.0 (3.5-5.0)* 4.0 (3.0-5.0)* 4.0 (3.0-4.0)* Simvastatin (n ⫽ 15) 2.0 (2.0-2.0)*† 2.0 (2.0-2.0)*† 2.0 (1.5-2.0)*† NOTE. Values are presented as median (interquartile range). The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; the simvastatin group received simvastatin orally for 5 days before the surgery. Aortic occlusion was performed in the control and simvastatin groups. Abbreviation: POD, postoperative day. *p ⬍ 0.05 compared with the sham group. †p ⬍ 0.05 compared with the control group.
The neuroprotective effect of statins also has been suggested in several studies.8,9,15,33 In particular, compared with other statins, simvastatin is more lipophilic and more permeable to the blood-spinal barrier, which could offer a more neuroprotective effect.9,34 The neuroprotective effects of simvastatin in in vivo models of the brain,7-9 peripheral nerve,35 and spinal cord16-19 also have been investigated; however, its results in a spinal cord mechanical injury model have been inconsistent. Han et al16 investigated the therapeutic efficacy of simvastatin treatment after spinal cord mechanical injury. In their study, simvastatin was administered orally 1 day after spinal cord mechanical injury and then daily for 5 weeks. They suggested that simvastatin treatment starting 1 day after spinal cord injury improves locomotor recovery. This neuroprotective effect may be related to the upregulation of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. However, Lee et al17 and Mann et al18 reported that simvastatin treatment after spinal cord mechanical injury failed to offer a neurologic benefit. They explained that the lack of neurologic benefits after simvastatin treatment was caused by the biomechanical severity of the mechanical injury and a “ceiling effect” on the locomotor recovery. The present study is in line with the study by Saito et al.19 They found that treatment with simvastatin, 10 mg/kg administered subcutaneously before ischemia and after ischemia, attenuated hind limb motor dysfunction and histopathologic changes in spinal cord ischemia-reperfusion injury for 48 hours. However, taking into consideration that most motor neurons were preserved until 2 days but were lost selectively at 7 days of reperfusion after transient spinal cord ischemia,36 the present study with a 1-week observation period clearly showed that pretreatment with simvastatin can decrease the neurologic deficit after spinal cord ischemia. Furthermore, the present study showed a distinctive pretreatment effect of simvastatin on spinal cord ischemia. For a spinal cord mechanical injury in a clinical setting, pretreatment is impossible. However, for thoracoabdominal aortic aneurysm surgery, pharmacologic pretreatment deserves consideration because it is possible to perform elective surgery and paraplegia remains a serious complication after spinal cord ischemia despite some strategies to protect the spinal cord, such as cerebrospinal fluid drainage37 and systemic hypothermia.38
In the present study, 1 dose (10 mg/kg/day) of simvastatin was administered orally for 5 days before ischemia. Previous studies have reported protective effects using much smaller doses of simvastatin (0.5 or 1 mg/kg),26,39,40 but in those experiments, simvastatin was administered intravenously,39 intraperitoneally,40 or subcutaneously.26 In the present study, the oral route of administration was chosen because of the clinical accessibility. With oral use, the absorbed fraction of simvastatin in the blood is 60% to 80% and the bioavailability is 5%.41 For the dosage of simvastatin, Urban et al8 and Hayashi et al9 showed that pretreatment with simvastatin, 20 mg/kg/day orally administered for 2 weeks before ischemia, significantly improved neurologic outcomes in rat cerebral ischemia models. Shabanzadeh et al42 suggested that treatment with simvastatin, 100 mg/kg/day for 2 weeks before a middle cerebral artery occlusion, decreased the neurologic injury in a rat stroke model. Furthermore, in a dose-ranging study, 3-day pretreatment with oral simvastatin at 10 mg/kg/day, but not at 2 mg/kg/day, significantly decreased the myocardial infarct size in an in vivo rat heart model.43 Slijper et al14 showed that oral simvastatin, 10 mg/kg given immediately before and 24 hours after surgery, prevented gut mucosal damage and inhibited apoptosis after intestinal ischemic injury in rats. In general, rats adopt to higher doses of simvastatin compared with other species, including humans.44 In humans, simvastatin is relatively safe at a normal dosage (maximal recommended human dose of simvastatin, 1 mg/kg/day; maximum dose approved by the Food and Drug
Fig 2. Number of normal motor neurons in the anterior horn of the spinal cord. The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; and the simvastatin received simvastatin orally for 5 days before the surgery. The aortic occlusion was performed in the control and simvastatin groups. Vertical bars represent the average number of normal motor neurons, and the error bars indicate 1 SD. The number of normal motor neurons in the anterior horn of the spinal cord (anterior to a line drawn through the central canal perpendicular to the vertebral axis) was counted in 3 sections for each animal and then averaged. Compared with the groups in which an aortic occlusion was performed, the number of normal motor neurons was significantly larger in the sham group. The simvastatin group had a larger number of motor neurons compared with the control group; *p < 0.05 compared with the sham group; †p < 0.05 compared with the control group.
SIMVASTATIN AND SPINAL CORD INJURY
83
Fig 3. Representative histologic findings of spinal cord sections stained with hematoxylin and eosin (original magnification, ⴛ200). (A) The sham group showed no specific histologic change. (B) Vacuolization and a significant loss of motor neuron cells were observed in the control group. (C) The simvastatin group showed a better preservation of motor neurons. (Color version of figure is available online.)
Administration, 80 mg/day); however, it has been is associated with increased hepatic enzyme and myopathy in a dose-dependent manner.45 In the present study, the authors chose the 10-mg/kg dose and a shorter administration schedule of simvastatin (for 5 days) compared with those in other experimental studies indicating a neuroprotective effect,8,9,42 considering their translation into human spinal cord ischemia-reperfusion injury. Further research is required to determine the optimal clinical dose considering risks and benefits. Compared with humans, although there are differences in the size and localization, rats possess a heterosegmental aorta with radicular arteries from 1 anterior artery and 2 posterior spinal arteries and the greater anterior radicular artery (Adamkiewicz artery) as the ventral feeder to the lower thoracic and lumbosacral cord. Furthermore, an additional blood supply may be derived from the branches of the 2 subclavian arteries, and a collateral flow exists down through the ventral chest wall and the abdominal free wall anastomosing with the iliac arteries.46,47 Therefore, anatomically, rats would appear to be one of the most suitable experimental spinal cord ischemia models for studying the pathophysiology, complications, and outcome after interventions. In the present study, the authors used a rat spinal cord ischemia model based on the method of Taira and Marsala.20 Taira and Marsala found that the collateral flow during aortic occlusion nearly was eliminated at a proximal arterial pressure of 40 mmHg. Thus, in subsequent researches using their rat spinal cord ischemia model,21,48,49 the proximal arterial pressure was maintained at 40 mmHg during the aortic occlusion. However, in the present study, the proximal arterial pressure during spinal cord ischemia was maintained at 80 mmHg because of the clinical practice. In clinical settings, mean arterial pressure has been recommended to be maintained at ⱖ80 mmHg to preserve spinal cord perfusion during aortic clamping.50 In addition, to induce a certain degree of motor deficit (not complete paraplegia) in the control group, the authors per-
formed pilot studies to determine the occlusion time with the proximal arterial pressure of 80 mmHg during spinal cord ischemia. Rats underwent aortic occlusion during increments of 15 seconds for 10 minutes (n ⫽ 6 in each group) at a proximal arterial pressure of 80 mmHg, and then the neurologic assessment was performed daily until 3 days after reperfusion. The present results showed that the group with 10 minutes 30 seconds of occlusion time displayed moderate motor deficits adequate for the authors’ experimental design. In the present study, isoflurane was used to maintain anesthesia. It has been suggested that isoflurane may be neuroprotective against spinal cord ischemia,51,52 and the effect that isoflurane may have had on the results cannot be excluded. However, isoflurane was used in all groups, so its effects on the results were controlled for. There are two limitations to this study. First, the authors tried to investigate the neuroprotective effect of simvastatin given for 5 days before a spinal cord ischemia-reperfusion insult and not to elucidate the mechanism of the protective effect. Further studies are needed to determine the mechanism of the neuroprotective effect of simvastatin. Second, the present study was performed in a rat experimental model. The rat spinal cord vasculature and collateral system are remarkably similar to the spinal vascular system in humans,46,47 which suggests that the rat model is appropriate for an experimental spinal cord ischemia model. However, it should not be excluded that there might be differences in the results according to the species. In conclusion, pretreatment with simvastatin significantly attenuated neurologic injury after a spinal cord ischemiareperfusion injury in a rat model. Taking into consideration the safety profile of simvastatin in clinical practice, the translational potential of simvastatin pretreatment is promising for ischemia-reperfusion injury in patients undergoing thoracoabdominal aortic surgery. For this, further studies on the optimal dosing and administration timing are required.
84
HWANG, HAN, AND HAN
REFERENCES 1. Svensson LG, Crawford ES, Hess KR, et al: Experience with 1509 patients undergoing thoracoabdominal aortic operations. J Vasc Surg 17:357-368, 1993 2. Marsala M, Sorkin LS, Yaksh TL: Transient spinal ischemia in rat: Characterization of spinal cord blood flow, extracellular amino acid release, and concurrent histopathological damage. J Cereb Blood Flow Metab 14:604-614, 1994 3. Lu K, Liang CL, Liliang PC, et al: Inhibition of extracellular signal-regulated kinases 1/2 provides neuroprotection in spinal cord ischemia/reperfusion injury in rats: Relationship with the nuclear factor-kappaB-regulated anti-apoptotic mechanisms. J Neurochem 114: 237-246, 2010 4. Yin R, Zhu J, Wang Z, et al: Simvastatin attenuates cardiac isograft ischemia-reperfusion injury by down-regulating CC chemokine receptor-2 expression. J Thorac Cardiovasc Surg 134:780-788, 2007 5. Di Napoli P, Antonio Taccardi A, Grilli A, et al: Simvastatin reduces reperfusion injury by modulating nitric oxide synthase expression: An ex vivo study in isolated working rat hearts. Cardiovasc Res 51:283-293, 2001 6. O’Driscoll G, Green D, Taylor RR: Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation 95:1126-1131, 1997 7. Balduini W, Mazzoni E, Carloni S, et al: Prophylactic but not delayed administration of simvastatin protects against long-lasting cognitive and morphological consequences of neonatal hypoxic-ischemic brain injury, reduces interleukin-1 and tumor necrosis factor-␣ mRNA induction, and does not affect endothelial nitric oxide synthase expression. Stroke 34:2007-2012, 2003 8. Urban P, Pavlíková M, Sivonová M, et al: Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemia/ reperfusion in rats: Effect of neuroprotectant simvastatin. Cell Mol Neurobiol 29:181-192, 2009 9. Hayashi T, Hamakawa K, Nagotani S, et al: HMG CoA Reductase Inhibitors reduce ischemic brain injury of Wistar rats through decreasing oxidative stress on neurons. Brain Res 1037:52-58, 2005 10. Bao N, Ushikoshi H, Kobayashi H, et al: Simvastatin reduces myocardial infarct size via increased nitric oxide production in normocholesterolemic rabbits. J Cardiol 53:102-107, 2009 11. Naidu BV, Woolley SM, Farivar AS, et al: Simvastatin ameliorates injury in an experimental model of lung ischemia-reperfusion. J Thorac Cardiovasc Surg 126:482-489, 2003 12. Lai IR, Chang KJ, Tsai HW, et al: Pharmacological preconditioning with simvastatin protects liver from ischemia-reperfusion injury by heme oxygenase-1 induction. Transplantation 85:732-738, 2008 13. Todorovic Z, Nesic Z, Stojanovic´ R, et al: Acute protective effects of simvastatin in the rat model of renal ischemia-reperfusion injury: It is never too late for the pretreatment. J Pharmacol Sci 107:465-470, 2008 14. Slijper N, Sukhotnik I, Chemodanov E, et al: Effect of simvastatin on intestinal recovery following gut ischemia-reperfusion injury in a rat. Pediatr Surg Int 26:105-110, 2010 15. Pannu R, Barbosa E, Singh AK, et al: Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats. J Neurosci Res 79:340-350, 2005 16. Han X, Yang N, Xu Y, et al: Simvastatin treatment improves functional recovery after experimental spinal cord injury by upregulating the expression of BDNF and GDNF. Neurosci Lett 487:255-259, 2011 17. Lee JH, Tigchelaar S, Liu J, et al: Lack of neuroprotective effects of simvastatin and minocycline in a model of cervical spinal cord injury. Exp Neurol 225:219-230, 2010
18. Mann CM, Lee JH, Hillyer J, et al: Lack of robust neurologic benefits with simvastatin or atorvastatin treatment after acute thoracic spinal cord contusion injury. Exp Neurol 221:285-295, 2010 19. Saito T, Tsuchida M, Umehara S, et al: Reduction of spinal cord ischemia/reperfusion injury with simvastatin in rats. Anesth Analg 113:565-571, 2011 20. Taira Y, Marsala M: Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathologic changes after increasing intervals of aortic occlusion in the rat. Stroke 27:18501858, 1996 21. Umehara S, Goyagi T, Nishikawa T, et al: Esmolol and landiolol, selective 1-adrenoreceptor antagonists, provide neuroprotection against spinal cord ischemia and reperfusion in rats. Anesth Analg 110:1133-1137, 2010 22. Ludman A, Venugopal V, Yellon DM, et al: Statins and cardioprotection—More than just lipid lowering? Pharmacol Ther 122:30-43, 2009 23. Istvan ES: Structural mechanism for statin inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Am Heart J 144:S27S32, 2002 24. Laufs U, La Fata V, Plutzky J, et al: Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97:1129-1135, 1998 25. Rikitake Y, Kawashima S, Takeshita S, et al: Anti-oxidative properties of fluvastatin, an HMG-CoA reductase inhibitor, contribute to prevention of atherosclerosis in cholesterol-fed rabbits. Atherosclerosis 154:87-96, 2001 26. Scalia R, Gooszen ME, Jones SP, et al: Simvastatin exerts both anti-inflammatory and cardioprotective effects in apolipoprotein E-deficient mice. Circulation 103:2598-2603, 2001 27. Weitz-Schmidt G, Welzenbach K, Brinkmann V, et al: Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 7:687-692, 2001 28. Pahan K, Sheikh FG, Namboodiri AM, et al: Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J Clin Invest 100:2671-2679, 1997 29. Yang YJ, Zhao JL, You SJ, et al: Post-infarction treatment with simvastatin reduces myocardial no-reflow by opening of the KATP channel. Eur J Heart Fail 9:30-36, 2007 30. Fonarow GC, Wright RS, Spencer FA, et al: Effect of statin use within the first 24 hours of admission for acute myocardial infarction on early morbidity and mortality. Am J Cardiol 96:611-616, 2005 31. Lenderink T, Boersma E, Gitt AK, et al: Patients using statin treatment within 24 h after admission for ST-elevation acute coronary syndromes had lower mortality than non-users: A report from the first Euro Heart Survey on acute coronary syndromes. Eur Heart J 27:17991804, 2006 32. Colivicchi F, Guido V, Tubaro M, et al: Effects of atorvastatin 80 mg daily early after onset of unstable angina pectoris or non-Q-wave myocardial infarction. Am J Cardiol 90:872-874, 2002 33. Ucak A, Onan B, Guler A, et al: Rosuvastatin, a new generation 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, reduces ischemia/reperfusion-induced spinal cord tissue injury in rats. Ann Vasc Surg 25:686-695, 2011 34. Sierra S, Ramos MC, Molina P, et al: Statins as neuroprotectants: A comparative in vitro study of lipophilicity, blood-brainbarrier penetration, lowering of brain cholesterol, and decrease of neuron cell death. J Alzheimers Dis 23:307-318, 2011 35. Gholami MR, Abolhassani F, Pasbakhsh P, et al: The effects of simvastatin on ischemia-reperfusion injury of sciatic nerve in adult rats. Eur J Pharmacol 590:111-114, 2008
SIMVASTATIN AND SPINAL CORD INJURY
36. Sakurai M, Hayashi T, Abe K, et al: Delayed and selective motor neuron death after transient spinal cord ischemia: A role of apoptosis? J Thorac Cardiovasc Surg 115:1310-1315, 1998 37. Fleck TM, Koinig H, Moidl R, et al: Improved outcome in thoracoabdominal aortic aneurysm repair: The role of cerebrospinal fluid drainage. Neurocrit Care 2:11-16, 2005 38. Kouchoukos NT, Daily BB, Rokkas CK, et al: Hypothermic bypass and circulatory arrest for operations on the descending thoracic and thoracoabdominal aorta. Ann Thorac Surg 60:67-76, 1995 39. Nesic´ Z, Todorovic´ Z, Stojanovic´ R, et al: Single-dose intravenous simvastatin treatment attenuates renal injury in an experimental model of ischemia-reperfusion in the rat. J Pharmacol Sci 102:413-417, 2006 40. Lefer DJ, Scalia R, Jones SP, et al: HMG-CoA reductase inhibition protects the diabetic myocardium from ischemia-reperfusion injury. FASEB J 15:1454-1456, 2001 41. Shitara Y, Sugiyama Y: Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions. Pharmacol Ther 112:71-105, 2006 42. Shabanzadeh AP, Shuaib A, Wang CX: Simvastatin reduced ischemic brain injury and perfusion deficits in an embolic model of stroke. Brain Res 1042:1-5, 2005 43. Birnbaum Y, Lin Y, Ye Y, et al: Pretreatment with high-dose statin, but not low-dose statin, ezetimibe, or the combination of lowdose statin and ezetimibe, limits infarct size in the rat. J Cardiovasc Pharmacol Ther 13:72-79, 2008
85
44. Gerson RJ, MacDonald JS, Alberts AW, et al: Animal safety and toxicology of simvastatin and related hydroxy-methylglutaryl-coenzyme A reductase inhibitors. Am J Med 87:28S-38S, 1989 45. de Lemos JA, Blazing MA, Wiviott SD, et al: Early intensive vs a delayed conservative simvastatin strategy in patients with acute coronary syndromes: Phase Z of the A to Z trial. JAMA 292:13071316, 2004 46. Tveten L: Spinal cord vascularity. III. The spinal cord arteries in man. Acta Radiol Diagn (Stockh) 17:257-273, 1976 47. Woollam DH, Millen JW: The arterial supply of the spinal cord and its significance. J Neurol Neurosurg Psychiatry 18:97-102, 1955 48. Kakinohana M, Fuchigami T, Nakamura S, et al: Intrathecal administration of morphine, but not small dose, induced spastic paraparesis after a noninjurious interval of aortic occlusion in rats. Anesth Analg 96:769-775, 2003 49. Horiuchi T, Kawaguchi M, Sakamoto T, et al: The effects of the delta-opioid agonist SNC80 on hind-limb motor function and neuronal injury after spinal cord ischemia in rats. Anesth Analg 99:235-240, 2004 50. Griepp RB, Griepp EB: Spinal cord perfusion and protection during descending thoracic and thoracoabdominal aortic surgery: The collateral network concept. Ann Thorac Surg 83:S865-S869, 2007 51. Park HP, Jeon YT, Hwang JW, et al: Isoflurane preconditioning protects motor neurons from spinal cord ischemia: Its dose-response effects and activation of mitochondrial adenosine triphosphate-dependent potassium channel. Neurosci Lett 387:90-94, 2005 52. Sang H, Cao L, Qiu P, et al: Isoflurane produces delayed preconditioning against spinal cord ischemic injury via release of free radicals in rabbits. Anesthesiology 105:953-960, 2006
significantly lower motor deficit index compared with the control group (2.0, 2.0-2.0, v 4.0, 3.5-5.0; p < 0.001). This trend was sustained at day 7 (2.0, 1.5-2.0, v 4.0, 3.0-4.0; p < 0.001). The simvastatin group displayed a significantly larger number of normal motor neurons compared with the control group (mean ⴞ SD, 31.7 ⴞ 6.1 v 20.4 ⴞ 4.4; p < 0.001). However, compared with the sham group, the simvastatin group displayed fewer intact motor neurons (sham group, 38.5 ⴞ 5.1; p ⴝ 0.005). Conclusions: Pretreatment with simvastatin, 10 mg/kg, given orally for 5 days before the ischemia-reperfusion insult, improved the neurologic outcome and preserved more normal motor neurons compared with the control group in a rat model of spinal cord ischemia-reperfusion. © 2012 Elsevier Inc. All rights reserved.
S
10 mg/kg (Zocor, Merck, Whitehouse Station, NJ) mixed with normal saline (1 mL) daily for 5 days before ischemia. Oral administration was performed by oral gavage using a 16-gauge feeding needle. Male Sprague-Dawley rats (300-350 g; n ⫽ 45) were anesthetized in an acryl box with isoflurane (5 vol%) in 100% oxygen. After induction, anesthesia was maintained using a facial mask for the inhalation of 1.0 to 2.5 vol% isoflurane driven by an oxygen flow of 2 L/min. Rats were placed in the supine position and the hair in the left inguinal area and neck was shaved. The tail artery was exposed and cannulated with a polyethylene catheter for heparin injection and distal arterial pressure monitoring. For the induction of spinal cord ischemia, the left femoral artery was exposed, and a 2Fr Fogarty catheter (Fogarty Arterial Embolectomy Catheter, Edwards Lifesciences, Irvine, CA) was inserted into the descending thoracic aorta so the tip of the catheter reached the left subclavian artery (11 cm from the site of insertion). The left carotid artery was exposed and cannulated with a 20-gauge catheter (BD Insyte, Becton Dickinson, Sandy, UT) to monitor the proximal arterial pressure. The catheter was connected to a saline-filled external blood reservoir to control the proximal arterial pressure to 80 mmHg during the aortic occlusion. The body temperature was monitored with a rectal probe inserted 6 cm into the rectum and maintained at 37.5 ⫾ 0.5°C with a heating blanket and overhead lamp. Spinal cord ischemia was induced by investigators who were blinded to the group assignment, using the method of Taira and Marsala20 (Fig 1). After completion of the cannulation, heparin (150 U) was injected into the tail artery and the balloon of the Fogarty catheter was inflated with saline, 0.05 mL. Simultaneously, the blood was drained from the carotid artery
PINAL CORD ISCHEMIA remains a devastating neurologic complication of thoracoabdominal aortic surgery. The incidence of paraparesis or paraplegia after thoracoabdominal aortic aneurysm repair has been reported to be 16%.1 The mechanism of paraplegia after thoracoabdominal aortic surgery has been considered an ischemia-reperfusion injury secondary to aortic clamping and declamping during the surgery. In ischemia-reperfusion injury of the spinal cord, the glutamate-mediated excitotoxicity,2 reactive oxygen species production, inflammation, and apoptosis3 play a key role in neuronal cell death. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), currently used as cholesterol-lowering agents, provide benefits other than lowering lipids, such as anti-inflammatory effects,4 antioxidants,5 and improved endothelial function.6 The beneficial effects of statins on ischemia-reperfusion injury have been reported in various organs, including the brain,7-9 heart,4,10 lungs,11 liver,12 kidneys13 and intestines.14 The effect of statins in mechanical spinal cord injury was reported first by Pannu et al15 who found that the administration of statin resulted in significant decreases of inflammatory cytokines and neuronal cell death and a significant improvement in locomotor scores. However, the potential for statins to promote neurologic recovery after spinal cord mechanical injury has been studied in only a limited number of reports,16-19 and the neurologic benefits are controversial. Furthermore, the pretreatment effect of simvastatin has not been determined clearly. This study was designed to evaluate the pretreatment effect of simvastatin on spinal cord ischemia-reperfusion injury. METHODS All animal experiments and animal care were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The following experimental protocol was approved by the institutional animal care and use committee of a university hospital. Rats were assigned randomly to 1 of 3 groups: (1) the sham group (n ⫽ 15) was administered oral saline (1 mL) daily for 5 days; (2) the control group (n ⫽ 15) was administered oral saline (1 mL) daily for 5 days; and (3) the simvastatin group (n ⫽ 15) received oral simvastatin
KEY WORDS: simvastatin, spinal cord ischemia, thoracoabdominal aortic surgery, paraplegia
From the ⴱDepartment of Anesthesiology and Pain Medicine, Seoul National University, Bundang Hospital, Seongnamsi, Gyeonggido, Korea; and †Department of Anesthesiology and Pain Medicine, School of Medicine, Ewha Womans University, Seoul, Korea. This study was supported by grant number 11-2010-018 from Seoul National University Bundang Hospital research fund. Address reprint requests to Jong In Han, MD, PhD, Department of Anesthesiology and Pain Medicine, School of Medicine, Ewha Womans University, 911, Mok-dong, Yangcheon-gu, Seoul, 158-710, Korea. E-mail: [email protected] © 2012 Elsevier Inc. All rights reserved. 1053-0770/2701-0001$36.00/0 doi:10.1053/j.jvca.2012.01.025
Journal of Cardiothoracic and Vascular Anesthesia, Vol 27, No 1 (February), 2013: pp 79-85
79
80
HWANG, HAN, AND HAN
stepping reflex), and the maximum deficit was indicated by a score of 6. The MDI was evaluated by an observer who was blinded to the group assignment. After the last neurologic assessment, the rats were anesthetized deeply with isoflurane and were perfused transcardially with heparinized saline, 100 mL. In brief, the heart was exposed, a 23-gauge needle was inserted into the left ventricle, and the right atrium was cut. Heparinized saline (100 mL) passed through the needle, circulated around the body, and exited through the open right atrium. The spinal cord was removed and fixed in 10% buffered formalin for 24 hours. Spinal cord segments at the L3-L5 level were embedded in paraffin. Transverse sections were cut at a thickness of 4 m and stained with hematoxylin and eosin. Neuronal injury was evaluated at ⫻200 magnification by an investigator blinded to the group assignment. In paraplegic animals, the anterior spinal cord was destroyed markedly and the disappearance of normal motor neurons was significant. To assess the degree of ischemic neuronal injury, the number of normal motor neurons in the anterior horn of the spinal cord (anterior to a line drawn through the central canal perpendicular to the vertebral axis) was counted in 3 sections for each animal and then averaged.21 SPSS 15 (SPSS Inc, Chicago, IL) was used for statistical analysis. Data were expressed as mean ⫾ SD or median (interquartile range). Physiologic data and the number of motor neurons were compared using a one-way analysis of variance followed by the Dunnett post hoc test. At each time point, the hind limb motor function of the 3 groups was compared with a Kruskal-Wallis test followed by a Mann-Whitney U test. The change in the MDI within each group was compared by the Friedman test followed by a Wilcoxon rank-sum test. Statistical significance was accepted when the p value was ⬍0.05. RESULTS
Fig 1. A diagram illustrating the preparation for spinal cord ischemia in a rat.
into the external reservoir to control the proximal arterial pressure at 80 mmHg by withdrawing blood during the aortic occlusion. The success of the aortic occlusion was confirmed by an immediate decrease and sustained loss of distal arterial pressure. The sham group was prepared in the same manner, but spinal cord ischemia was not induced. After 10 minutes 30 seconds of aortic occlusion, the Fogarty balloon was deflated, and the drained blood was reinfused. After the reperfusion, all catheters were removed and the wounds were closed. The rats were allowed to recover from anesthesia and were returned to their cages. Mean arterial pressure, heart rate, and body temperature were monitored continuously. Values were recorded before the aortic occlusion, 5 minutes after the aortic occlusion, and 10 minutes after reperfusion. Arterial blood gas and hematocrit levels were measured before the aortic occlusion and 10 minutes after reperfusion. Neurologic function was assessed daily until 7 days after reperfusion. The motor function of the hind limb was evaluated by an assessment of the ambulation and placing/stepping reflex according to the method of Taira and Marsala20 (Table 1). The placing/stepping reflex was assessed by dragging the dorsum of the hind paw over the edge of a surface. Normally, this evokes a coordinating lifting and placing/ stepping response. The motor deficit index (MDI) was defined as the sum of the scores (ambulation with lower extremities plus placing/
Mean distal arterial pressure, heart rate, and rectal temperature were similar among the 3 groups at each time point (Table 2). The arterial pH and partial pressures of carbon dioxide and oxygen were not different among the groups at any time point. There was no difference in hematocrit values among the groups before the aortic occlusion. After reperfusion, the control and simvastatin groups showed significantly decreased values of hematocrit compared with the sham group (p ⫽ 0.003, each group v sham group), but there was no difference between the control and simvastatin groups (Table 3). All animals survived until the final neurologic assessment at 7 days after reperfusion. The sham group showed an MDI of 0 throughout the experimental period. On day 1 after reperfusion, the simvastatin group showed a significantly lower MDI com-
Table 1. Assessment of Ambulation and Placing/Stepping Reflex Ambulation (Walking With Lower Extremities)
0 ⫽ normal (symmetric and coordinated ambulation) 1 ⫽ toes flat under body when walking but ataxia present 2 ⫽ knuckle walking 3 ⫽ movement in lower extremities but unable to knuckle walk 4 ⫽ no movement, drags lower extremities
Placing/Stepping Reflex
0 ⫽ normal
1 ⫽ weak 2 ⫽ no stepping
SIMVASTATIN AND SPINAL CORD INJURY
81
Table 2. Hemodynamic Variables and Temperature
Before aortic occlusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15) During aortic occlusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15) After reperfusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15)
MDAP (mm Hg)
HR (beats/min)
Temperature (°C)
99.1 ⫾ 7.6 100.1 ⫾ 11.1 100.0 ⫾ 8.5
313.5 ⫾ 17.7 318.5 ⫾ 22.0 303.3 ⫾ 24.8
37.4 ⫾ 0.2 37.3 ⫾ 0.3 37.5 ⫾ 0.3
No occlusion 6.2 ⫾ 1.3 6.0 ⫾ 1.1
No occlusion 301.1 ⫾ 15.3 290.1 ⫾ 29.7
No occlusion 37.4 ⫾ 0.3 37.6 ⫾ 0.3
103.2 ⫾ 7.4 99.1 ⫾ 14.9 102.5 ⫾ 13.3
315.4 ⫾ 14.8 320.7 ⫾ 21.5 296.8 ⫾ 28.4
37.4 ⫾ 0.2 37.5 ⫾ 0.3 37.5 ⫾ 0.3
NOTE. Values are presented as mean ⫾ SD. The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; the simvastatin group received simvastatin orally for 5 days before the surgery. Aortic occlusion was performed in the control and simvastatin groups. The groups did not show any difference in the measured hemodynamic variables at each time point. Abbreviations: HR, heart rate; MDAP, mean distal arterial pressure.
pared with the control group. This trend was sustained through day 7. Within each group, there was no statistically significant change in the MDI during the study period (Table 4). The number of normal motor neurons in the anterior spinal cord is presented in Fig 2. The simvastatin group displayed a significantly larger number of normal motor neurons compared with the control group (31.7 ⫾ 6.1 v 20.4 ⫾ 4.4, p ⬍ 0.001). However, compared with the sham group, the simvastatin group displayed fewer intact motor neurons (sham group, 38.5 ⫾ 5.1; p ⫽ 0.005). Representative photographs from each group are shown in Fig 3. DISCUSSION
The present study showed that pretreatment with simvastatin 10 mg/kg given orally for 5 days before an ischemia-reperfusion insult improved the neurologic outcome and preserved more normal motor neurons compared with the control group in a rat model of spinal cord ischemia-reperfusion. Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been shown convincingly to produce effects
that are dependent on and independent of lipid lowering.22 They inhibit the conversion of acetyl and acetoacetyl coenzyme A to mevalonate in the formation of cholesterol and prevent the formation of isoprenoids, which are critical proteins in the inflammatory cascade.23 The lipid-lowering independent effects of statins include some beneficial effects on endothelial function and anti-inflammatory actions. Statins increase endothelial nitric oxide production and improve endothelial function by directly upregulating nitric oxide synthase expression24 and decreasing oxidative stress.25 Furthermore, they attenuate leukocyte-endothelial cell interactions26,27 and inhibit the expression of proinflammatory cytokines, including tumor necrosis factor-␣, interleukin-1, and interleukin-6.28 The protective effects of statins have been reported in experimental heart,4,10,29 intestine,14 liver12 and kidney13 ischemia-reperfusion models. In particular, the protective effects of statins have been demonstrated well in the heart. The use of statin therapy for acute myocardial ischemia was related to a significantly lower rate of early complications and in-hospital mortality in human clinical studies.30-32
Table 3. Hematologic Parameters
Before aortic occlusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15) After reperfusion Sham (n ⫽ 15) Control (n ⫽ 15) Simvastatin (n ⫽ 15)
pH
PaO2 (mmHg)
PaCO2 (mmHg)
Hematocrit (%)
7.40 ⫾ 0.02 7.40 ⫾ 0.02 7.39 ⫾ 0.02
259.1 ⫾ 47.5 252.4 ⫾ 39.2 222.0 ⫾ 31.1
35.6 ⫾ 2.2 36.1 ⫾ 3.1 35.4 ⫾ 2.3
39.1 ⫾ 2.9 39.3 ⫾ 2.9 39.4 ⫾ 2.4
7.38 ⫾ 0.02 7.35 ⫾ 0.06 7.35 ⫾ 0.04
269.8 ⫾ 34.4 263.3 ⫾ 29.6 231.7 ⫾ 36.5
35.1 ⫾ 2.4 35.9 ⫾ 3.4 36.1 ⫾ 3.2
39.3 ⫾ 2.2 36.2 ⫾ 2.1* 36.8 ⫾ 2.2*
NOTE. Values are presented as mean ⫾ SD. The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; the simvastatin group received simvastatin orally for 5 days before the surgery. Aortic occlusion was performed in the control and simvastatin groups. The groups did not show any difference in blood gas data at each time point. Hematocrit did not differ among the 3 groups before aortic occlusion; after reperfusion, the control and simvastatin groups showed significantly lower hematocrit values compared with the sham group. Abbreviations: PaCO2, partial arterial pressure of carbon dioxide; PaO2, partial arterial pressure of oxygen. *p ⬍ 0.05 compared with the sham group.
82
HWANG, HAN, AND HAN
Table 4. Motor Deficit Index POD 1
POD 3
POD 7
Sham (n ⫽ 15) 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0) Control (n ⫽ 15) 4.0 (3.5-5.0)* 4.0 (3.0-5.0)* 4.0 (3.0-4.0)* Simvastatin (n ⫽ 15) 2.0 (2.0-2.0)*† 2.0 (2.0-2.0)*† 2.0 (1.5-2.0)*† NOTE. Values are presented as median (interquartile range). The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; the simvastatin group received simvastatin orally for 5 days before the surgery. Aortic occlusion was performed in the control and simvastatin groups. Abbreviation: POD, postoperative day. *p ⬍ 0.05 compared with the sham group. †p ⬍ 0.05 compared with the control group.
The neuroprotective effect of statins also has been suggested in several studies.8,9,15,33 In particular, compared with other statins, simvastatin is more lipophilic and more permeable to the blood-spinal barrier, which could offer a more neuroprotective effect.9,34 The neuroprotective effects of simvastatin in in vivo models of the brain,7-9 peripheral nerve,35 and spinal cord16-19 also have been investigated; however, its results in a spinal cord mechanical injury model have been inconsistent. Han et al16 investigated the therapeutic efficacy of simvastatin treatment after spinal cord mechanical injury. In their study, simvastatin was administered orally 1 day after spinal cord mechanical injury and then daily for 5 weeks. They suggested that simvastatin treatment starting 1 day after spinal cord injury improves locomotor recovery. This neuroprotective effect may be related to the upregulation of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. However, Lee et al17 and Mann et al18 reported that simvastatin treatment after spinal cord mechanical injury failed to offer a neurologic benefit. They explained that the lack of neurologic benefits after simvastatin treatment was caused by the biomechanical severity of the mechanical injury and a “ceiling effect” on the locomotor recovery. The present study is in line with the study by Saito et al.19 They found that treatment with simvastatin, 10 mg/kg administered subcutaneously before ischemia and after ischemia, attenuated hind limb motor dysfunction and histopathologic changes in spinal cord ischemia-reperfusion injury for 48 hours. However, taking into consideration that most motor neurons were preserved until 2 days but were lost selectively at 7 days of reperfusion after transient spinal cord ischemia,36 the present study with a 1-week observation period clearly showed that pretreatment with simvastatin can decrease the neurologic deficit after spinal cord ischemia. Furthermore, the present study showed a distinctive pretreatment effect of simvastatin on spinal cord ischemia. For a spinal cord mechanical injury in a clinical setting, pretreatment is impossible. However, for thoracoabdominal aortic aneurysm surgery, pharmacologic pretreatment deserves consideration because it is possible to perform elective surgery and paraplegia remains a serious complication after spinal cord ischemia despite some strategies to protect the spinal cord, such as cerebrospinal fluid drainage37 and systemic hypothermia.38
In the present study, 1 dose (10 mg/kg/day) of simvastatin was administered orally for 5 days before ischemia. Previous studies have reported protective effects using much smaller doses of simvastatin (0.5 or 1 mg/kg),26,39,40 but in those experiments, simvastatin was administered intravenously,39 intraperitoneally,40 or subcutaneously.26 In the present study, the oral route of administration was chosen because of the clinical accessibility. With oral use, the absorbed fraction of simvastatin in the blood is 60% to 80% and the bioavailability is 5%.41 For the dosage of simvastatin, Urban et al8 and Hayashi et al9 showed that pretreatment with simvastatin, 20 mg/kg/day orally administered for 2 weeks before ischemia, significantly improved neurologic outcomes in rat cerebral ischemia models. Shabanzadeh et al42 suggested that treatment with simvastatin, 100 mg/kg/day for 2 weeks before a middle cerebral artery occlusion, decreased the neurologic injury in a rat stroke model. Furthermore, in a dose-ranging study, 3-day pretreatment with oral simvastatin at 10 mg/kg/day, but not at 2 mg/kg/day, significantly decreased the myocardial infarct size in an in vivo rat heart model.43 Slijper et al14 showed that oral simvastatin, 10 mg/kg given immediately before and 24 hours after surgery, prevented gut mucosal damage and inhibited apoptosis after intestinal ischemic injury in rats. In general, rats adopt to higher doses of simvastatin compared with other species, including humans.44 In humans, simvastatin is relatively safe at a normal dosage (maximal recommended human dose of simvastatin, 1 mg/kg/day; maximum dose approved by the Food and Drug
Fig 2. Number of normal motor neurons in the anterior horn of the spinal cord. The sham group received saline orally for 5 days before the surgery; the control group received saline orally for 5 days before the surgery; and the simvastatin received simvastatin orally for 5 days before the surgery. The aortic occlusion was performed in the control and simvastatin groups. Vertical bars represent the average number of normal motor neurons, and the error bars indicate 1 SD. The number of normal motor neurons in the anterior horn of the spinal cord (anterior to a line drawn through the central canal perpendicular to the vertebral axis) was counted in 3 sections for each animal and then averaged. Compared with the groups in which an aortic occlusion was performed, the number of normal motor neurons was significantly larger in the sham group. The simvastatin group had a larger number of motor neurons compared with the control group; *p < 0.05 compared with the sham group; †p < 0.05 compared with the control group.
SIMVASTATIN AND SPINAL CORD INJURY
83
Fig 3. Representative histologic findings of spinal cord sections stained with hematoxylin and eosin (original magnification, ⴛ200). (A) The sham group showed no specific histologic change. (B) Vacuolization and a significant loss of motor neuron cells were observed in the control group. (C) The simvastatin group showed a better preservation of motor neurons. (Color version of figure is available online.)
Administration, 80 mg/day); however, it has been is associated with increased hepatic enzyme and myopathy in a dose-dependent manner.45 In the present study, the authors chose the 10-mg/kg dose and a shorter administration schedule of simvastatin (for 5 days) compared with those in other experimental studies indicating a neuroprotective effect,8,9,42 considering their translation into human spinal cord ischemia-reperfusion injury. Further research is required to determine the optimal clinical dose considering risks and benefits. Compared with humans, although there are differences in the size and localization, rats possess a heterosegmental aorta with radicular arteries from 1 anterior artery and 2 posterior spinal arteries and the greater anterior radicular artery (Adamkiewicz artery) as the ventral feeder to the lower thoracic and lumbosacral cord. Furthermore, an additional blood supply may be derived from the branches of the 2 subclavian arteries, and a collateral flow exists down through the ventral chest wall and the abdominal free wall anastomosing with the iliac arteries.46,47 Therefore, anatomically, rats would appear to be one of the most suitable experimental spinal cord ischemia models for studying the pathophysiology, complications, and outcome after interventions. In the present study, the authors used a rat spinal cord ischemia model based on the method of Taira and Marsala.20 Taira and Marsala found that the collateral flow during aortic occlusion nearly was eliminated at a proximal arterial pressure of 40 mmHg. Thus, in subsequent researches using their rat spinal cord ischemia model,21,48,49 the proximal arterial pressure was maintained at 40 mmHg during the aortic occlusion. However, in the present study, the proximal arterial pressure during spinal cord ischemia was maintained at 80 mmHg because of the clinical practice. In clinical settings, mean arterial pressure has been recommended to be maintained at ⱖ80 mmHg to preserve spinal cord perfusion during aortic clamping.50 In addition, to induce a certain degree of motor deficit (not complete paraplegia) in the control group, the authors per-
formed pilot studies to determine the occlusion time with the proximal arterial pressure of 80 mmHg during spinal cord ischemia. Rats underwent aortic occlusion during increments of 15 seconds for 10 minutes (n ⫽ 6 in each group) at a proximal arterial pressure of 80 mmHg, and then the neurologic assessment was performed daily until 3 days after reperfusion. The present results showed that the group with 10 minutes 30 seconds of occlusion time displayed moderate motor deficits adequate for the authors’ experimental design. In the present study, isoflurane was used to maintain anesthesia. It has been suggested that isoflurane may be neuroprotective against spinal cord ischemia,51,52 and the effect that isoflurane may have had on the results cannot be excluded. However, isoflurane was used in all groups, so its effects on the results were controlled for. There are two limitations to this study. First, the authors tried to investigate the neuroprotective effect of simvastatin given for 5 days before a spinal cord ischemia-reperfusion insult and not to elucidate the mechanism of the protective effect. Further studies are needed to determine the mechanism of the neuroprotective effect of simvastatin. Second, the present study was performed in a rat experimental model. The rat spinal cord vasculature and collateral system are remarkably similar to the spinal vascular system in humans,46,47 which suggests that the rat model is appropriate for an experimental spinal cord ischemia model. However, it should not be excluded that there might be differences in the results according to the species. In conclusion, pretreatment with simvastatin significantly attenuated neurologic injury after a spinal cord ischemiareperfusion injury in a rat model. Taking into consideration the safety profile of simvastatin in clinical practice, the translational potential of simvastatin pretreatment is promising for ischemia-reperfusion injury in patients undergoing thoracoabdominal aortic surgery. For this, further studies on the optimal dosing and administration timing are required.
84
HWANG, HAN, AND HAN
REFERENCES 1. Svensson LG, Crawford ES, Hess KR, et al: Experience with 1509 patients undergoing thoracoabdominal aortic operations. J Vasc Surg 17:357-368, 1993 2. Marsala M, Sorkin LS, Yaksh TL: Transient spinal ischemia in rat: Characterization of spinal cord blood flow, extracellular amino acid release, and concurrent histopathological damage. J Cereb Blood Flow Metab 14:604-614, 1994 3. Lu K, Liang CL, Liliang PC, et al: Inhibition of extracellular signal-regulated kinases 1/2 provides neuroprotection in spinal cord ischemia/reperfusion injury in rats: Relationship with the nuclear factor-kappaB-regulated anti-apoptotic mechanisms. J Neurochem 114: 237-246, 2010 4. Yin R, Zhu J, Wang Z, et al: Simvastatin attenuates cardiac isograft ischemia-reperfusion injury by down-regulating CC chemokine receptor-2 expression. J Thorac Cardiovasc Surg 134:780-788, 2007 5. Di Napoli P, Antonio Taccardi A, Grilli A, et al: Simvastatin reduces reperfusion injury by modulating nitric oxide synthase expression: An ex vivo study in isolated working rat hearts. Cardiovasc Res 51:283-293, 2001 6. O’Driscoll G, Green D, Taylor RR: Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation 95:1126-1131, 1997 7. Balduini W, Mazzoni E, Carloni S, et al: Prophylactic but not delayed administration of simvastatin protects against long-lasting cognitive and morphological consequences of neonatal hypoxic-ischemic brain injury, reduces interleukin-1 and tumor necrosis factor-␣ mRNA induction, and does not affect endothelial nitric oxide synthase expression. Stroke 34:2007-2012, 2003 8. Urban P, Pavlíková M, Sivonová M, et al: Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemia/ reperfusion in rats: Effect of neuroprotectant simvastatin. Cell Mol Neurobiol 29:181-192, 2009 9. Hayashi T, Hamakawa K, Nagotani S, et al: HMG CoA Reductase Inhibitors reduce ischemic brain injury of Wistar rats through decreasing oxidative stress on neurons. Brain Res 1037:52-58, 2005 10. Bao N, Ushikoshi H, Kobayashi H, et al: Simvastatin reduces myocardial infarct size via increased nitric oxide production in normocholesterolemic rabbits. J Cardiol 53:102-107, 2009 11. Naidu BV, Woolley SM, Farivar AS, et al: Simvastatin ameliorates injury in an experimental model of lung ischemia-reperfusion. J Thorac Cardiovasc Surg 126:482-489, 2003 12. Lai IR, Chang KJ, Tsai HW, et al: Pharmacological preconditioning with simvastatin protects liver from ischemia-reperfusion injury by heme oxygenase-1 induction. Transplantation 85:732-738, 2008 13. Todorovic Z, Nesic Z, Stojanovic´ R, et al: Acute protective effects of simvastatin in the rat model of renal ischemia-reperfusion injury: It is never too late for the pretreatment. J Pharmacol Sci 107:465-470, 2008 14. Slijper N, Sukhotnik I, Chemodanov E, et al: Effect of simvastatin on intestinal recovery following gut ischemia-reperfusion injury in a rat. Pediatr Surg Int 26:105-110, 2010 15. Pannu R, Barbosa E, Singh AK, et al: Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats. J Neurosci Res 79:340-350, 2005 16. Han X, Yang N, Xu Y, et al: Simvastatin treatment improves functional recovery after experimental spinal cord injury by upregulating the expression of BDNF and GDNF. Neurosci Lett 487:255-259, 2011 17. Lee JH, Tigchelaar S, Liu J, et al: Lack of neuroprotective effects of simvastatin and minocycline in a model of cervical spinal cord injury. Exp Neurol 225:219-230, 2010
18. Mann CM, Lee JH, Hillyer J, et al: Lack of robust neurologic benefits with simvastatin or atorvastatin treatment after acute thoracic spinal cord contusion injury. Exp Neurol 221:285-295, 2010 19. Saito T, Tsuchida M, Umehara S, et al: Reduction of spinal cord ischemia/reperfusion injury with simvastatin in rats. Anesth Analg 113:565-571, 2011 20. Taira Y, Marsala M: Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathologic changes after increasing intervals of aortic occlusion in the rat. Stroke 27:18501858, 1996 21. Umehara S, Goyagi T, Nishikawa T, et al: Esmolol and landiolol, selective 1-adrenoreceptor antagonists, provide neuroprotection against spinal cord ischemia and reperfusion in rats. Anesth Analg 110:1133-1137, 2010 22. Ludman A, Venugopal V, Yellon DM, et al: Statins and cardioprotection—More than just lipid lowering? Pharmacol Ther 122:30-43, 2009 23. Istvan ES: Structural mechanism for statin inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Am Heart J 144:S27S32, 2002 24. Laufs U, La Fata V, Plutzky J, et al: Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97:1129-1135, 1998 25. Rikitake Y, Kawashima S, Takeshita S, et al: Anti-oxidative properties of fluvastatin, an HMG-CoA reductase inhibitor, contribute to prevention of atherosclerosis in cholesterol-fed rabbits. Atherosclerosis 154:87-96, 2001 26. Scalia R, Gooszen ME, Jones SP, et al: Simvastatin exerts both anti-inflammatory and cardioprotective effects in apolipoprotein E-deficient mice. Circulation 103:2598-2603, 2001 27. Weitz-Schmidt G, Welzenbach K, Brinkmann V, et al: Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 7:687-692, 2001 28. Pahan K, Sheikh FG, Namboodiri AM, et al: Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J Clin Invest 100:2671-2679, 1997 29. Yang YJ, Zhao JL, You SJ, et al: Post-infarction treatment with simvastatin reduces myocardial no-reflow by opening of the KATP channel. Eur J Heart Fail 9:30-36, 2007 30. Fonarow GC, Wright RS, Spencer FA, et al: Effect of statin use within the first 24 hours of admission for acute myocardial infarction on early morbidity and mortality. Am J Cardiol 96:611-616, 2005 31. Lenderink T, Boersma E, Gitt AK, et al: Patients using statin treatment within 24 h after admission for ST-elevation acute coronary syndromes had lower mortality than non-users: A report from the first Euro Heart Survey on acute coronary syndromes. Eur Heart J 27:17991804, 2006 32. Colivicchi F, Guido V, Tubaro M, et al: Effects of atorvastatin 80 mg daily early after onset of unstable angina pectoris or non-Q-wave myocardial infarction. Am J Cardiol 90:872-874, 2002 33. Ucak A, Onan B, Guler A, et al: Rosuvastatin, a new generation 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, reduces ischemia/reperfusion-induced spinal cord tissue injury in rats. Ann Vasc Surg 25:686-695, 2011 34. Sierra S, Ramos MC, Molina P, et al: Statins as neuroprotectants: A comparative in vitro study of lipophilicity, blood-brainbarrier penetration, lowering of brain cholesterol, and decrease of neuron cell death. J Alzheimers Dis 23:307-318, 2011 35. Gholami MR, Abolhassani F, Pasbakhsh P, et al: The effects of simvastatin on ischemia-reperfusion injury of sciatic nerve in adult rats. Eur J Pharmacol 590:111-114, 2008
SIMVASTATIN AND SPINAL CORD INJURY
36. Sakurai M, Hayashi T, Abe K, et al: Delayed and selective motor neuron death after transient spinal cord ischemia: A role of apoptosis? J Thorac Cardiovasc Surg 115:1310-1315, 1998 37. Fleck TM, Koinig H, Moidl R, et al: Improved outcome in thoracoabdominal aortic aneurysm repair: The role of cerebrospinal fluid drainage. Neurocrit Care 2:11-16, 2005 38. Kouchoukos NT, Daily BB, Rokkas CK, et al: Hypothermic bypass and circulatory arrest for operations on the descending thoracic and thoracoabdominal aorta. Ann Thorac Surg 60:67-76, 1995 39. Nesic´ Z, Todorovic´ Z, Stojanovic´ R, et al: Single-dose intravenous simvastatin treatment attenuates renal injury in an experimental model of ischemia-reperfusion in the rat. J Pharmacol Sci 102:413-417, 2006 40. Lefer DJ, Scalia R, Jones SP, et al: HMG-CoA reductase inhibition protects the diabetic myocardium from ischemia-reperfusion injury. FASEB J 15:1454-1456, 2001 41. Shitara Y, Sugiyama Y: Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions. Pharmacol Ther 112:71-105, 2006 42. Shabanzadeh AP, Shuaib A, Wang CX: Simvastatin reduced ischemic brain injury and perfusion deficits in an embolic model of stroke. Brain Res 1042:1-5, 2005 43. Birnbaum Y, Lin Y, Ye Y, et al: Pretreatment with high-dose statin, but not low-dose statin, ezetimibe, or the combination of lowdose statin and ezetimibe, limits infarct size in the rat. J Cardiovasc Pharmacol Ther 13:72-79, 2008
85
44. Gerson RJ, MacDonald JS, Alberts AW, et al: Animal safety and toxicology of simvastatin and related hydroxy-methylglutaryl-coenzyme A reductase inhibitors. Am J Med 87:28S-38S, 1989 45. de Lemos JA, Blazing MA, Wiviott SD, et al: Early intensive vs a delayed conservative simvastatin strategy in patients with acute coronary syndromes: Phase Z of the A to Z trial. JAMA 292:13071316, 2004 46. Tveten L: Spinal cord vascularity. III. The spinal cord arteries in man. Acta Radiol Diagn (Stockh) 17:257-273, 1976 47. Woollam DH, Millen JW: The arterial supply of the spinal cord and its significance. J Neurol Neurosurg Psychiatry 18:97-102, 1955 48. Kakinohana M, Fuchigami T, Nakamura S, et al: Intrathecal administration of morphine, but not small dose, induced spastic paraparesis after a noninjurious interval of aortic occlusion in rats. Anesth Analg 96:769-775, 2003 49. Horiuchi T, Kawaguchi M, Sakamoto T, et al: The effects of the delta-opioid agonist SNC80 on hind-limb motor function and neuronal injury after spinal cord ischemia in rats. Anesth Analg 99:235-240, 2004 50. Griepp RB, Griepp EB: Spinal cord perfusion and protection during descending thoracic and thoracoabdominal aortic surgery: The collateral network concept. Ann Thorac Surg 83:S865-S869, 2007 51. Park HP, Jeon YT, Hwang JW, et al: Isoflurane preconditioning protects motor neurons from spinal cord ischemia: Its dose-response effects and activation of mitochondrial adenosine triphosphate-dependent potassium channel. Neurosci Lett 387:90-94, 2005 52. Sang H, Cao L, Qiu P, et al: Isoflurane produces delayed preconditioning against spinal cord ischemic injury via release of free radicals in rabbits. Anesthesiology 105:953-960, 2006