Journal of Clinical Neuroscience 17 (2010) 490494

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Journal of Clinical Neuroscience

journal homepage: www.elsevier.com/locate/jocn

Laboratory Study

The effectiveness of dexmedetomidine in experimental spinal cord injury compared to methylprednisolone in rats
Sanser Gul a,*, Volkan Hanci b, Burak Bahadir c, Serefden Acikgoz d, Sibel Bektas c, Handan Ankarali e, Murat Kalayci a, Bektas Acikgoz a
a

Department of Neurosurgery, School of Medicine, Zonguldak Karaelmas University, Kozlu, Zonguldak 67600, Turkey Department of Anaesthesiology, School of Medicine, Zonguldak Karaelmas University, Zonguldak, Turkey Department of Pathology, School of Medicine, Zonguldak Karaelmas University, Zonguldak, Turkey d Department of Biochemistry, School of Medicine, Zonguldak Karaelmas University, Zonguldak, Turkey e Department of Biostatistics, School of Medicine, Zonguldak Karaelmas University, Zonguldak, Turkey
b c

a r t i c l e

i n f o

a b s t r a c t
The present study aimed to investigate the neuroprotective efcacy of dexmedetomidine in a rat experimental spinal cord injury model. The rats (n = 40) were equally divided into four groups: G1, G2, G3, and G4. Rats in the G1 group underwent a laminectomy only. For the rats in the G2, G3, and G4 groups, spinal cord injury was induced by placing an aneurysm clip extradurally for 60 s at T10. The rats in G2 did not receive any post-injury treatment. Immediately after trauma was induced, rats in G3 were given methylprednisolone (30 mg/kg) and in G4, dexmedetomidine (10 lg/kg), both intraperitoneally. The rats were sacriced under anesthesia 24 hours later and 1.5 cm lengths of injured spinal cord were obtained. Malonyldialdehyde values were signicantly increased in G2 compared to G1, G3 and G4 (p < 0.05). The neuronal cell count in G1 was signicantly higher than in G2 and G3 (p = 0.0001; p = 0.007). G4 had higher cell counts compared to G2 and G3 (p = 0.0001; p = 0.05). These ndings indicated that dexmedetomidine might have neuroprotective effects in spinal cord injury. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 28 February 2009 Accepted 17 May 2009

Keywords: Dexmedetomidine Methylprednisolone Spinal cord injury

1. Introduction Spinal cord injury affects individuals at any age and may cause permanent neurologic decits as well as secondary complications.1,2 Because there is generally a low probability of full neurological recovery after spinal cord injury, posttraumatic biochemical and pathological processes that worsen functional loss or secondary autodestructive mechanisms caused by trauma have attracted much interest.36 Although the exact mechanism is unknown, many pathological changes, including edema, inammation, altered blood ow, and changes in microvascular permeability may contribute to the development of secondary injury by various pathways that include free oxygen radicals and lipid peroxidation.26 Therefore, many studies have focused on neuroprotective agents against secondary injury.39 Methylprednisolone has been extensively studied in both experimental and clinical trials and has become the most commonly used agent as a standard care for spinal cord injury.3,4,8,1012 However, in recent years, the use of corticosteroids has become controversial.1317

Dexmedetomidine is a potent and selective alpha-2 adrenergic agonist. Considering that norepinephrin release may be a component of cerebral injury, dexmedetomidine may potentially provide neuroprotection.7,1821 Dexmedetomidine reduces ischemic damage, possibly by attenuating excessive release of norepinephrin.19,20 However, studies on central nervous system trauma, particularly those involving the spinal cord, are rare.2 The aim of this study was to investigate the neuroprotective effect of dexmedetomidine compared to methylprednisolone in rats with spinal cord injury.

2. Materials and methods Approval was obtained from the Zonguldak Karaelmas University Ethics Committee. Forty male adult WistarAlbino rats weighing 230250 g were included in the study. The animal study was performed at the Experimental Surgery, Research and Animal Laboratory. All rats were maintained at 2225 C and ambient humidity and were supplied with sufcient food and uids and were mostly on a 12-hour light/dark cycle before and after surgery. The 40 rats were divided equally into four groups (n = 10): G1, G2, G3, and G4. Rats in the control group (G1) underwent a laminectomy only. In G2, G3, and G4, spinal cord injury was induced

* Corresponding author. Tel.: +90 3722612001/3196; fax: +90 3722610264. E-mail address: drsanser@yahoo.com (S. Gul). 0967-5868/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2009.05.041

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by placing an aneurysm clip extradurally at T10. Immediately after trauma had been induced, the rats in G3 and G4 received single intraperitoneal doses of 30 mg/kg methylprednisolone (G3; Mustafa Nevzat, Istanbul, Turkey) and 10 lg/kg dexmedetomidine hydrochloride (G4; Hospira, Rocky Mount, NC, USA). Anesthesia was induced by intraperitoneal administration of ketamine hydrochloride at 60 mg/kg. Rats were placed prone on the operating table. The area from T9 to L3 was isolated and disinfected. The skin was incised at the midline, the fascia was cut open, paravertebral muscles were removed subperiostally, and a laminectomy was performed at T10. Injury was applied to the spinal cord by the clip method, using an aneurysm clip for 60 s (Yasargil FE 750, Valley, Aesculap, PA, USA) (Fig. 1). Neurologic results were observed at 12 hours and 24 hours after spinal cord injury in all rats by: (i) gross neurologic examination pinching the rats tail to cause pain; and (ii) Rivlin and Tators inclined plane test,22 which measured the maximum angle at which the rats, on a at platform, could maintain a stationary position for at least 5 minutes. Paraplegia was identied in all rats by applying a painful stimulus to the tail. Rats were sacriced 24 hours after spinal cord injury using a lethal dose of ketamine hydrochloride. The operated area was carefully dissected, and a 1.5-cm long sample of spinal cord was obtained; the rostral 0.5 cm was used for biochemical analysis and the caudal 1 cm for histological examination. For biochemical analysis, tissue samples were rinsed with physiologic saline and frozen just after sampling and stored at 20 C; blood samples were also obtained. The biochemical data obtained were the concentrations of malonyldialdehyde (MDA) in spinal cord tissue and paraoxonase (PON) and high-density lipoprotein cholesterol (HDL-C) levels in the serum. The blood samples collected at the time of death were centrifuged at 1000 g for 10 minutes at 4 C to separate the plasma. Aliquots were transferred into polyethylene tubes and stored at 80 C. All tissue was washed twice with cold saline solution, placed into glass bottles, labeled and stored at 80 C until processing. After cutting the cord tissue into small pieces with scissors, the sample was homogenized in 10 volumes of 150 mM ice-cold KCl for 2 minutes at 5000 revolutions per minute using a glass polytetrauoroethylene homogenizer (Ultra Turrax T18 Basic, IKA Works, Wilmington, NC, USA). The homogenate was then centrifuged at 5000 g for 15 minutes, and the resulting supernatant was analysed. High-performance liquid chromatographic (HPLC) analysis was performed using a Shimadzu HPLC system (Kyoto, Japan) with an MDA kit (Immundiagnostik,

Bensheim, Germany). PON-1 activity was measured according to Gan et al., with paraoxon as substrate, in the presence of 1 mM Ca2+ in 100 mM TrisHCl buffer (pH 8.0) using a Shimadzu UV-1601 spectrophotometer (Kyoto, Japan). HDL-C levels were measured using commercially available kits with an automated analyzer (Hitachi Modular P800, Tokyo, Japan).23 For histopathological investigation, spinal cord samples were xed in neutral buffered 10% formalin and stored at 4 C for 1 week and subsequently placed in fresh xative. Fixed tissue samples were embedded in parafn and horizontally sectioned into 5 lm slices with a microtome. Three sections were obtained from each specimen: (i) from the center of the damaged cord segment, (ii) 3 mm rostral to the centre; and (iii) 3 mm caudal to the centre. After deparafnization, sections were mounted on slides and stained with hematoxylin and eosin (H&E). The slides were assessed under a light microscope for edema, congestion, hemorrhage, neuronal degeneration, and neuronal loss. Sections stained with cresyl violet from the center of the injured cord segments were also used to assess neuronal loss using an image analysis program (Leica QWINPlus v. 3.1.0; Leica, Solms, Germany). Ten microscope elds from gray matter at 100 magnication were randomly selected, and the neuronal cell count was determined (cell counts/mm2). Statistical analysis was computed using the Statistical Package for the Social Sciences version 10.0 program (SPSS, Chicago, IL, USA). All results are given as the mean standard error (SE). A one-way analysis of variance (ANOVA) and post-hoc Tukey tests were used to obtain MDA and PON values as well as neuronal cell counts. The level of statistical signicance was set at p < 0.05. An alternative KruskalWallis test was also used for conrmation when high standard errors were obtained. The correlation between MDA values and neuronal cell counts was determined using the Pearson correlation analysis. 3. Results Statistical results from both the one-way ANOVA and the alternative KruskalWallis test revealed similar ndings. MDA, PON and HDL values are given in Table 1. MDA values in the G2 group were higher than in G1, G3, and G4 (p < 0.05), and there was no statistical correlation between MDA concentrations of G1 (53.2 3.14), G3 (47.3 3.4), and G4 (68.1 10.6). There was no signicant difference in mean PON and HDL values between G1, G2, G3, and G4. Histological examination of spinal cord tissue of G1 rats revealed normal ndings (Fig. 2A). In G2 rats, edema and hemorrhage was accompanied by prominent neuronal degeneration in the gray matter (Fig. 2B). Samples from G3 showed edema and prominent hemorrhage with neuronal degeneration (Fig. 2C). In G4, modest histopathological changes included mild congestion and edema (Fig. 2D). Table 2 and Fig. 3 show mean neuronal cell counts. The mean neuronal cell count in G1 rats is higher than in G2 (p = 0.0001) and G3 (p = 0.007). G4 also had higher cell counts compared to G2 (p = 0.0001) and G3 (p = 0.05). However, there were no signi-

Table 1 Concentrations (mean standard error) of tissue MDA, serum PON, and serum HDL in the spinal cord of rats exposed to experimental spinal cord injury Group No. 1 2 3 4 Fig. 1. Intraoperative photograph showing spinal cord compression at T10 by application of an aneurysm clip. This gure is available in colour at www.science direct.com. n 10 10 10 10 MDA (nmol/g tissue) 53.2 3.14* 118.2 20.9 47.3 3.4* 68.1 10.6* PON (U/L) 34.3 7.8 20.6 4.5 37.8 6.6 26.6 4.2 HDL (mg/dL) 44.7 1.7 52.1 1.7 47.2 3.8 50.7 2.2

HDL = high density lipoprotein, MDA = malonyldialdehyde, PON = paraoxonase. p < 0.05, compared to group 2.

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Fig. 2. Representative photomicrographs of spinal cord sections showing: (A) control group (G1) with nearly normal histological features demonstrating evenly spaced wellpreserved neurons (haematoxylin and eosin [H&E] 100); (B) trauma group (G2) with edema, hemorrhage, and neuronal degeneration in the gray matter (H&E 200) (inset, neuronal loss demonstrated by cresyl violet stain 200); (C) methylprednisolone-treated group (G3) with hemorrhage associated with degeneration, and loss of neurons (H&E 200); (D) dexmedetomidine-treated group (G4) with prominent reduction in degenerative changes and prevention of neuronal loss (H&E 200) (inset, neuronal loss demonstrated by cresyl violet stain 400).

Mean +- 2 SE of neuronal cell counts

Table 2 Neuronal cell counts (neurons/mm2) (mean standard error) in the spinal cord of rats exposed to experimental spinal cord injury: control (G1), trauma (G2), methylprednisolone (G3), and dexmedetomidine (G4) groups Group No. 1 2 3 4 n 10 10 10 10 Cell count 27.7 1.5 14.1 0.92 19.4 1.5 25.8 2.4

30

25

G1 > G2 (p = 0.0001); G1 > G3 (p = 0.007); G4 > G2 (p = 0.0001); G4 > G3 (p = 0.05). There were no signicant differences between G1 and G4 (p = 0.856) or between G2 and G3 (p = 0.137).

20

cant differences between G1 and G4 rats (p = 0.856) or between G2 and G3 rats (p = 0.137). 4. Discussion Spinal cord injury results in a high rate of morbidity and mortality.14,810 Following a primary injury that disrupts the cord integrity, a secondary form of injury occurs as a result of cell death and tissue damage due to cellular and biochemical processes, which include free radical formation and lipid peroxidation.1,3,4,8,9 Although the exact mechanism remains unknown, many pathologic changes including edema, inammation, altered blood ow and changes in microvascular permeability, as well as sympathetic stimuli inducing norepinephrine, may also contribute to the development of secondary injury.1,2,7 Based on the results of the third National Acute Spinal Cord Injury Study randomized controlled trial, methylprednisolone has been widely used for the treatment of spinal cord injuries.11,12

15

Group 1

Group 2
2

Group 3

Group 4

Fig. 3. Number of neurons (neurons/mm ) in the gray matter of the spinal cord of rats exposed to experimental spinal cord injury: control group (G1), trauma (G2), methylprednisolone (G3), and dexmedetomidine (G4) groups showing that the number of neurons was the same for G1 and G4 rats (p = 0.856) and for G2 and G3 rats (p = 0.137). (G1 > G2 [p = 0.0001]; G1 > G3 [p = 0.007]; G4 > G2 [p = 0.0001]; G4 > G3 [p = 0.05]).

Although effective in experimental and clinical studies,1,3,4,8,11,12 the use of high-dose corticosteroids has recently been questioned.1,1317 Therefore, an increasing number of studies now focus on potential neuroprotective agents against secondary injury.3,4,6,8,9 Anesthetic agents including etomidate,24 thiopental,

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and propofol25 may provide neuroprotection by preventing neuronal loss secondary to cord injury. Dexmedetomidine is a highly selective potent alpha2-adrenergic agonist increasingly used as an adjunct in general anesthesia.26 Considering that norepinephrin release may be a component of central nervous system injury, dexmedetomidine may potentially provide neuroprotection against this.7,1821 Many experimental studies have reported that dexmedetomidine provides neuronal protection against cell death by preventing ischemia and by delaying the development of infarction.5,7,8,21,2730 Decreased sympathetic tonus due to inhibition of norepinephrin release via presynaptic alpha2-adrenoreceptors or N-methyl-Daspartate antagonism have been classically attributed as the neuroprotective effects of dexmedetomidine.19,20,31,32 Dexmedetomidine may produce these effects by altering early gene expression in injured cells.18 Engelhard et al. proposed that dexmedetomidine prevents apoptotic processes induced by incomplete cerebral ischemia/reperfusion and upregulates anti-apoptotic proteins Bcl2 and Mdm-2.31 In contrast, there are still some reports asserting that dexmedetomidine has no effect on neuronal damage.33 The effective dose level of dexmedetomidine against secondary injury without producing adverse side effects is also controversial. Low doses (36.5 lg/kg) are more effective and provide maximal inhibition of delayed neuronal death.7,18,21,32 In a study by Aslan et al., however, a lower dose of dexmedetomidine at 1 lg/kg failed to prevent apoptosis or neurodecits after traumatic spinal cord injury (2). In other studies, the neuroprotective effect of dexmedetomidine has been demonstrated at higher doses (up to 100 lg/kg).5,19,28,29,31 Jolkkonen et al. found that a dose of 9 lg/kg dexmedetomidine was sufcient for neuroprotection and did not produce any serious systemic adverse side effects.19 MDA is an end product and a sensitive indicator of lipid peroxidation produced by reactive oxygen species that results in damage to cell membranes. It correlates well with the degree of lipid peroxidation and secondary injury in models of experimental spinal cord injury.3,4 PON is a HDL-associated enzyme with antioxidant effects, and tissue PON levels can be used in monitoring the antioxidant defence system in experimental spinal cord injury models.34 In the current study, the protective effect of dexmedetomidine on traumatic spinal cord injury has been evaluated. Although some studies have evaluated the neuroprotective property of dexmedetomidine in the cerebrum,7,18,21,30 only two studies to our knowledge concern spinal cord injury.2,35 A dose of 10 lg/kg was chosen because it is presumed that lower doses might be ineffective and higher doses would incite adverse effects.5,19,28 We found that MDA levels were signicantly lower both in the methylprednisolone-treated (G3) and the dexmedetomidine-treated (G4) treated groups compared to the trauma group (G2). These results suggested that dexmedetomidine as well as methylprednisolone reduced MDA levels. Methylprednisolone treatment tended to decrease MDA levels by more than by dexmedetomidine but this was not signicant. PON levels tended to be higher in G3 and G4 groups than in G2, although no signicant difference was found between G1, G2, G3 and G4 groups. The non-signicant differences in PON levels may, to a certain extent, be explained by this enzyme possibly acting through different pathways. However, when histopathologic ndings and neuronal cell counts were considered, the cell counts were higher in the dexmedetomidine-treated group (G4) than in the trauma group (G2). Improved cell counts achieved by dexmedetomidine treatment (G4) also conrmed its neuroprotective potential, a nding supported by previous reports.7 However, methylprednisolone appeared to have no positive effect on neuronal cell count, in contrast to its potentially benecial effects on MDA levels. This observation may, to some extent, be explained by the presence of extensive hemorrhage seen in histological sections. As previously stated, corticosteroid treatment in spinal cord injury is controversial.

5. Conclusion The current study indicates that dexmedetomidine may have a neuroprotective effect in spinal cord injury, and its effects may be partially attributed to inhibition of lipid peroxidation. In addition, methylprednisolone seems to reduce lipid peroxidation, but its use is questionable because of the lack of histological correlation. Therefore, detailed experimental studies of dexmedetomidine are required to determine its neuroprotective effects and elucidate its exact mechanism of action. References
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