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Neuroprotective effects of racemic ketamine and (S)-ketamine on spinal cord injury in rat
Injury, Int. J. Care Injured 43 (2012) 1124–1130
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Neuroprotective effects of racemic ketamine and (S)-ketamine on spinal cord injury in rat Emine Arzu Kose a,*, Bulent Bakar b, Sebnem Kupana Ayva c, Kamer Kilinc d, Alpaslan Apan a a
Kirikkale University, School of Medicine, Department of Anaestesiology and Reanimation, Kirikkale, Turkey Kirikkale University, School of Medicine, Department of Neurosurgery, Kirikkale, Turkey c Kirikkale University, School of Medicine, Department of Pathology, Kirikkale, Turkey d Hacettepe University, School of Medicine, Department of Biochemistry, Ankara, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 29 February 2012
Background: The aim of this study was to investigate and to compare the potential neuroprotective effects of racemic ketamine, (S)-ketamine and methylprednisolone after an experimental spinal cord injury model in rats. Methods: Fifty-nine Wistar albino rats were divided into three main groups as acute stage (A), subacute stage (SA) and sham groups and then acute and subacute stage groups were divided into four groups regarding the used drug as control (CONT), racemic ketamine (RK), (S)-ketamine (SK) and methylprednisolone (MP) groups. A dorsal laminectomy was performed; and spinal cord injury was induced by using a temporary aneurysm clip. Four hours later from the clip compression, except those of the sham and control groups, the drugs (60 mg/kg racemic ketamine, 60 mg/kg (S)-ketamine or 30 mg/kg methylprednisolone) were administered intraperitoneally. At 72th h and 7th days of the study, the spinal cords of rats were removed from T8 level to the conus medullaris level. The specimens were and evaluated histopathologically, tissue lipid peroxidation (LPO) and myeloperoxidation (MPO) levels were measured and biochemically. Results: The histopathological results were similar both in the acute and in the subacute stage groups. There was a statistically significant difference among all groups regarding the tissue LPO levels (p < 0.001). There was a statistically significant difference between the CONT-A group and the MP-A, RKA and SK-A groups (p = 0.004, p < 0.001 and p = 0.007, respectively) in acute stage and between the CONT-SA group and SK-SA group (p = 0.002) in subacute stage. There was a statistically significant difference among all groups regarding the tissue MPO levels (p = 0.001). The median MPO levels were similar among acute stage groups (p = 0.057), but there was a statistical difference among subacute stage groups (p = 0.046). Conclusion: (S)-ketamine is more effective than methylprednisolone and racemic ketamine to reduce the LPO levels in subacute stage of spinal cord injury in rats. And, it is as effective as methylprednisolone in preventing secondary spinal cord injury histopathologically. ß 2012 Elsevier Ltd. All rights reserved.
Keywords: Racemic ketamine (S)-ketamine Methylprednisolone Spinal cord injury Rat
Introduction Traumatic spinal cord injury (SCI) is still a major clinical problem with permanent neurological deficits and secondary complications. The initial mechanical damage causing immediate cell death in the spinal cord is known as primary injury and inevitable. After primary injury, lesions greatly enlarge and worsen by secondary injury.1 Secondary injury mechanisms involve
* Corresponding author at: Kirikkale University, School of Medicine, Department of Anaestesiology and Reanimation, 71100-Kirikkale, Turkey. Tel.: +90 318 225 24 85/22 63; fax: +90 318 225 28 19. E-mail address: [email protected] (E.A. Kose). 0020–1383/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2012.02.022
excessive release of glutamate and aspartate, intracellular calcium overload, the activation of arachidonic acid cascade, and the induction of free radical induced lipid peroxidation (LPO) which has often been suggested to be an important factor in posttraumatic neuronal degeneration.2 Although the exact mechanism is unknown, altered blood flow and changes in microvascular permeability, as well as sympathetic stimuli including norepinephrine may contribute to the development of secondary injury.3–8 Methylprednisolone, a potent immunosupressive glucocorticoid, has beneficial effects in improving neurologic recovery when administered within 8 h after the onset of the SCI. It reduces the progression of oedema and has antioxidant and cell membrane stabilising properties.9 Taoka et al. showed that methylprednisolone
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reduces the severity of SCI by inhibiting activated leucocytes.10 The neuroprotective effect of methylprednisolone can be related to an interaction with the cytokine cascade and decrease in the production TNF-a and IL-6.11 Although the precise mechanism of action of methylprednisolone is not completely understood, it is the most commonly used agent as a standard care for SCI.12,13 While it is effective in experimental and clinical studies, the use of high-dose corticosteroids has recently been questioned.14,15 Therefore, an increasing number of studies now focus on potential neuroprotective agents against secondary injury.3–8 Ketamine is an anaesthetic agent, which inhibits N-methyl-Daspartate (NMDA) receptors. Ketamine has two enantiomers because of its molecular structure: S(+) ketamine and R() ketamine.16 Several experimental studies suggest that ketamine may be capable of producing neuroprotective effects.17–21 Racemic ketamine inhibits nitric oxide synthesis and nitric oxide-dependent cyclic guanosine monophosphate production stimulated by glutamate and glutamate analogues in primary culturs of cortical neurons and glia.19 This ketamine-induced inhibition of glutamate receptors may contribute to cellular protection against ischemic injury. The data obtained from experimental studies emphasize that the salutary actions of ketamine in models of neuronal injury appear to be stereoselective.17,18,21,22 While racemic and (S)- but not (R)-ketamine attenuate injury after glutamate exposure or axonal transection in rat hippocampal neurons in vitro, neuroregenerative effects appear only with (S)-ketamine.17 Ketamine may also decrease the severity of neuronal damage by interfering with the inflammatory response to ischemia. Ketamine suppresses lipopolysaccharide-induced TNF-a, IL-6, and IL-8 production and inhibits neutrophil adhesion to the endothelium in vitro.23 Ketamine also enhances neurologic outcome concomitant with reduction in plasma catecholamine concentrations in a rat model of incomplete cerebral ischemia.24 Additionally, it has been shown that, ketamine protects various tissues from ischemia/reperfusion (I/R) injury, such as myocardium, skeletal muscle, intestinal tissue, kidney and reduces malondialdehyde levels, a specific marker of LPO, in these tissues.25–28 To the best of our knowledge, there has been no research on the neuroprotective effects of ketamine-enantiomers after SCI. This study was designed to investigate and to compare the possible neuroprotective effects of racemic ketamine, (S)-ketamine and methylprednisolone after an experimental SCI model in rats.
Materials and methods Materials The investigation was conducted in accordance with the Guide for Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and approval has been received from the Animal Ethics Committee of The Ministry of Health Ankara Research and Training Hospital. Racemic ketamine (Ketalar1, Pfizer Inc., USA), (S)-ketamine (Ketanest1 S, Pfizer Inc., USA), and methylprednisolone (Depomedrol1, Pharmacia & Upjohn Company, Kalamazoo, USA) were used in this study. Fifty-nine male, Wistar albino rats weighing 250–300 g firstly were divided into three main groups as acute stage (A), subacute stage (SA) and sham groups; and then except the sham group which was not performed spinal cord injury, each main group was divided into four groups according to used study drug using a random numbers table. The acute stage study groups were named as following: - SHAM group (no SCI + no drug; n = 5).
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- CONT-A group (SCI + 1 mL % 0.9 NaCl intraperitoneal (i.p.); n = 6). - MP-A group (SCI + 30 mg/kg methylprednisolone in 1 mL volume i.p., followed by a maintenance dose of 5.4 mg/kg/h; n = 7). - RK-A group (SCI + 60 mg/kg racemic ketamine in 1 mL volume i.p.; n = 7). - SK-A group (SCI + 60 mg/kg (S)-ketamine in 1 mL volume i.p.; n = 7). The acute stage study groups were named as following: - CONT-SA group (SCI + 1 mL % 0.9 NaCl intraperitoneally i.p.; n = 6). - MP-SA group (SCI + 30 mg/kg methylprednisolone in 1 mL volume i.p., followed by a maintenance dose of 5.4 mg/kg/h; n = 7). - RK-SA group (SCI + 60 mg/kg racemic ketamine in 1 mL volume i.p.; n = 7). - SK-SA group (SCI + 60 mg/kg (S)-ketamine in 1 mL volume i.p.; n = 7). Methods All rats were anaesthetized via intramuscular injection of 30 mg/kg thiopental sodium (Penthal Sodyum, IE Ulugay Ilac San., I˙stanbul, Turkey) and 5 mg/kg xylazine HCl (Rompun1 %2, Bayer HealthCare AG, Germany); breathing was continued spontaneously with room air. Rats were positioned prone on operating table and a midline dorsal incision was done under a sterile technique. A dorsal laminectomy at thoracal 9–10 level was performed, and the dura mater was left intact. The spinal cord was exposed; and except SHAM group spinal cord injury was induced by using a temporary aneurysm clip (Mizuho1 Aneurysm Clip, Mizuho, Japan) by using a technique described before by Rivlin and Tator.29 The clip was removed after 60 s and the development of haemorrhagic contusion was seen at the point on the spinal cord where the clip was placed (Fig. 1) and then paravertebral fascia and skin were sutured with silk stitches. Four hours later from the clip compression, the study drugs were administered to rats via intraperitoneal injection using a 22G needle according to their included groups. In this study, methylprednisolone was administered in the doses of used in standard care protocol of SCI.12,13 As a maintenance dose, MP-A and MP-SA groups were given a total of 5.4 mg/kg/h intraperitoneal methylprednisolone treatment at 6 h intervals, with the total dose being 23 h. Additionally, due to the fact that Church et al. have shown that 60 mg/kg ketamine has neuroprotective effects in transient cerebral ischemia in rats, 60 mg/kg dose of ketamine was preferred in this study.30 After the administration of the study drugs, all rats were recovered from anaesthesia spontaneously under the blanket. Hind limb locomotor deficit resulted from SCI was observed in all rats which were clip compression was used. Seventy two hours later, all animals selected for acute stage groups; and 7 days later the animals selected for subacute stage groups were re-anaesthetized with intramuscular thiopenthal sodium and xylazine HCl and cardiac air embolization was performed for scarification. Dorsal incision of the animals was re-opened, and the spinal cord was totally removed from T8 level to the conus medullaris level. The spinal cord was divided into two parts horizontally. The proximal side included contusion lesion was stored in 10% buffered formaldehyde solution for histopathological examination at room temperature and the distal part was stored at 30 8C at dry air for biochemical examination. Specimen analysis For histopathological examination, all tissue samples were fixed at 10% buffered formaldehyde and processed according to
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ammonium bromide (HETAB) and 5 mM EDTA. The resulting suspension was centrifuged at 5000 g for 2 min and the supernatant was used for the activity measurement. The MPO activity was measured in a final volume of 1 mL containing 80 mM phosphate buffer (pH: 5.4), 0.5% HETAB, 1.6 mM synthetic substrate tetramethylbenzidine (TMB) initially dissolved in dimethylformamide, 2 mM H2O2 and the sample. The reaction was started at 37 8C by the addition of H2O2. The initial rate of MPO-catalyzed TMB oxidation was followed by recording the increase of absorbance at 655 nm (Shimadzu1 UV-120-02 spectrophotometry). The MPO activity was expressed as the amount of the enzyme producing one absorbance change per minute under assay conditions. Tissue-associated MPO activity was calculated as units per gram of wet tissue. Statistical analysis
Fig. 1. A dorsal laminectomy at thoracal 9–10 level was performed and traumatic spinal cord injury was induced by using a temporary aneurysm clip (a) exposed for 60 s (b).
Data were analysed using the SPSS 11.5 (SPSS Inc., Software, Chicago, Illinois, USA) statistical software. Tissue LPO levels were normally distributed and the variation was homogenous among all groups. Therefore, LPO levels were analyzed by using one-way analysis of variance (ANOVA) and with Tukey test so as to determine comparison among groups, and differences among groups, respectively. MPO levels were not normally distributed and the variation was not homogenous among all groups. So, MPO levels were analysed by using the Krusskal–Wallis test, and with the Mann–Whitney U test (with Bonferroni correction) so as to determine comparison among groups, and differences among groups, respectively. Histopathological data were analysed by using the Chi-Square test both to determine comparison among groups or differences among groups. p < 0.05 was considered statistically significant. Results
routine light microscopic tissue processing technique. Serial sections of 5 mm stained with haematoxylin–eosin were examined and photographed by a microscope (Leica1 Microsystems, Wetzlar GmbH). Each section was evaluated by experienced histopathologist blinded to the groups, and study drugs. For evaluation of the SCI histopathologically, a grading system described by Black et al. was used31 on all specimens as following: Grade 0: no destruction on the spinal cord tissue histopathologically. Grade I: mild neural tissue destruction and polymorphonuclear cell infiltration without neuronal cell lost; the posterior column of the spinal cord was affected Grade II: moderate neural tissue destruction and macrophage and/or histiocyte infiltration with white matter loss and central cavitation Grade III: severe neural tissue destruction with white and grey matter cystic necrosis and gliosis. Biochemical determinations were carried out by biochemist blinded to the animal groups, and test materials. Frozen tissue samples were weighted and homogenized in 1:10 (w:v) potassium phosphate buffer (50 mM, pH: 7.4) by using a dounce homogenizer. Thiobarbituric acid reactive substances (TBARS) were measured as an index of LPO by the method of Mihara et al.32,33 Tissue levels of lipid peroxides (as TBARS) were calculated as nanomole per gram wet tissue. Tissue-associated myeloperoxidation (MPO) activity was measured by the modified method of Suzuki et al.34,35 Tissue homogenate (0.5 mL) was centrifuged at 10,000 g for five minutes, and the pellet was resuspended in equal volume (0.5 mL) of 50 mM phosphate buffer (pH: 6.0) containing 0.5% hexadecyltrimethyl
Histopathological analysis Spinal cord sections from the sham group in 72 h after operation had normal histological structure (Fig. 2). There was a statistically significant difference among all groups regarding histopathological grades (X2 = 73.493, p < 0.001), and this significance was result from the SHAM group. There was a statistically significant difference neither among acute stage groups nor among the subacute stage groups regarding the histopathological grades (X2 = 6.396, p = 0.380; and X2 = 2.250, p = 0.522, respectively). The histopathological grades assigned for each group and the light microscopic appearance for each histopathological grade was shown in Table 1 and Fig. 2, respectively. Biochemical analysis There was a statistically significant difference among all groups regarding the tissue LPO levels (F = 6.591, p < 0.001), and this statistically significant difference was also observed either among acute stage groups (F = 12.150, p < 0.001) or among the subacute stage groups (F = 5.813, p = 0.004). There was a statistically significant difference between the CONT-A/MP-A, CONT-A/RK-A and CONT-A/SK-A groups (p = 0.004, p < 0.001 and p = 0.007, respectively) in acute stage (Fig. 3). When posthoc comparisons were made, no statistically difference was observed between the CONT-SA/MP-SA, and the CONT-SA/RK-SA groups (p = 0.062 and p = 0.061, respectively) but there was a statistically difference between the CONT-SA group and SK-SA group (p = 0.002) in subacute stage (Fig. 4). Mean LPO levels of the SHAM group were also statistically different from the CONT-A group (p < 0.001) and the CONT-SA group (p = 0.007) (Figs. 3 and 4).
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Fig. 2. Histopathological grades: Grade 0 = no neural tissue destruction. Grade I = mild neural tissue destruction and polymorphonuclear cell infiltration without neuronal cell lost; the posterior column of the spinal cord was affected. Grade II = moderate neural tissue destruction and macrophage and/or histiocyte infiltration with white matter lost and central cavitation. Grade III = severe neural tissue destruction with white and grey matter cystic necrosis and gliosis (H&E100, and H&E400).
There was a statistically significant difference among all groups regarding the tissue MPO levels (X2 = 27.259, p = 0.001). Nevertheless, when paired comparisons were made, no statistical significance was determined between compared groups. The median MPO levels were not statistically different among acute stage groups (X2 = 7.539, p = 0.057), but there was a statistical difference among subacute stage groups (X2 = 7.981, p = 0.046). However, no statistical significant difference was determined by paired comparisons. The median MPO levels for each group were shown in Table 2. Additionally, there was a statistically significant difference between the SHAM group and each of the other study groups except the RK-A group (p = 0.050) regarding the tissue MPO levels, when paired comparisons were made and Bonferroni correction was performed (Fig. 5). Discussion
Additionally, these results were in favour of (S)-ketamine in preventing neuronal damage in acute stage of SCI. When subacute stage groups were investigated, moderate to severe inflammatory reaction containing macrophages and histiocytes was observed. So, it can be said that none of the study drugs could reduce or completely block the macrophage and histiocyte infiltration into the injured neuronal tissue in the subacute stage of SCI. Although, there was not a statistical significance among subacute stage groups, the frequency of the Grade III degeneration was the lowest with a 14.3% frequency in SK-SA group; and it was higher in the MP-SA group than RK-SA group with 42.9% and 28.6% frequencies, respectively. On the base of these results, it can be suggested that, (S)-ketamine has a trend towards to be more effective agent than both methyprednisolone and racemic ketamine in preventing neuronal damage in subacute stage of SCI in rat as well as in acute stage and the beneficial effects of ketamine are stereoselective.17–21
Histopathological evaluation Biochemical evaluation In this study, there was mild to severe degree of neuronal destruction with demyelination and cavity formation in all acute stage groups except the SHAM group (Fig. 2). Grade III degeneration was lowest with a 14.3% frequency in SK-A group (Table 1). Because, there was not a statistical significance among acute stage groups regarding the histopathological grades, it can be said that racemic ketamine and (S)-ketamine are as effective as methyprednisolone to reduce the severity of neuronal damage by interfering with the inflammatory response to ischemia.11,23
Lipid peroxidation initiated by oxidative stress is one of the most important and destroying effects of ‘‘the free radicals following SCI. SCI disturbs the ability of mitochondria to carry out cellular respiration and oxidative phosphorylation. Ischemia also challenges tissue energy demands and function of active ion channels and cells then switch from aerobic to anaerobic metabolism and lead to cell death.36 When the LPO levels were evaluated in acute stage groups it was seen that there was a
Table 1 The histopathological grades for each group. Group Acute stage
Grade
Grade Grade Grade Grade
0 I II III
Subacute stage
SHAM
CONT
MP
RK
SK
CONT
MP
RK
SK
5(100) 0(0) 0(0) 0(0)
0(0) 1(16.7) 2(33.3) 3(50.0)
0(0) 0(0) 4(57.1) 3(42.9)
0(0) 0(0) 4(57.1) 3(42.9)
0(0) 0(0) 6(85.7) 1(14.3)
0(0) 0(0) 3(50.0) 3(50.0)
0(0) 0(0) 4(57.1) 3(42.9)
0(0) 0(0) 5(71.4) 2(28.6)
0(0) 0(0) 6(85.7) 1(14.3)
Data are expressed as numbers, with percentiles in parenthesis. CONT = control; MP = methylprednisolone; RK = racemic ketamine; SK = (S)-ketamine.
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Fig. 3. Tissue lipid peroxidation levels in the acute stage groups. CONT-A = control; MP-A = methylprednisolone; RK-A = racemic ketamine; SK-A = (S)-ketamine groups of acute stage; a = significant difference from the CONT-A group; b = significant difference from the SHAM group.
Fig. 5. Tissue myeloperoxidation levels in the acute and subacute stage groups. CONT-A = control; MP-A = methylprednisolone; RK-A = racemic ketamine; SKA = (S)-ketamine groups of acute stage; CONT-SA = control; MPSA = methylprednisolone; RK-SA = racemic ketamine; SK-SA = (S)-ketamine groups of subacute stage; a = significant difference from the SHAM group.
statistically significant difference between the CONT-A/MP-A, CONT-A/RK-A, and CONT-A/SK-A groups; but there was no statistical difference between the SHAM group and the study groups which were study drugs was given (Fig. 3). So, it can be said that all of the study drugs could reduce the LPO levels and both racemic ketamine and (S)-ketamine are as effective as methylprednisolone in acute stage of SCI in rat. This beneficial effect of ketamine can be explained by different mechanisms. Ketamine, a NMDA receptor antagonist, blockades an ion channel that is permeable to Ca++ and glutamate release by ischemic neurons. The activation of these receptors increases Ca++ influx and rapidly initiates cell necrosis and apoptosis. Thus, the blockade of NMDA receptors may potentially inhibits this excitotoxic injury.17,18 It has been shown that ketamine inhibits nitric oxide synthesis and nitric oxide-dependent cyclic guanosine monophosphate production stimulated by glutamate and glutamate analogues (e.g., NMDA, quisqualate, kainite) in primary culturs of cortical neurons and glia.19 This ketamine-induced inhibition of glutamate receptors
may contribute cellular protection against ischemic injury. Ketamine may also decrease the severity of neuronal damage by interfering with the inflammatory response to ischemia by suppressing lipopolysaccharide-induced TNF-a, IL-6, and IL-8 production and inhibiting neutrophil adhesion to the endothelium.23 When the LPO levels in subacute stage groups were evaluated, it was seen that the only statistical difference was between the CONT-SA group and SK-SA group, and there was no statistical difference between the SHAM group and the SK-SA group (Fig. 4). Thus, it can be said that among the study drugs only (S)-ketamine has a beneficial effect which prolongs to subacute stage. Neutrophils and other phagocytes produce hypochloride, a strong oxidant when the enzyme myeloperoxidase acts on hydrogen peroxide and chloride ion. Myeloperoxidase is a specific enzyme in large quantities in granules of the neutrophils and MPO activity is correlated with the absolute number of the neutrophils and their activations. Neutrophils infiltration into the injured spinal cord tissue occurs in 6 h and is followed by activated macrophages and microglia. It peaks in 24–48 h and may persist during the first week.37 In this study, there was a statistically significant difference between the SHAM group and the acute stage groups except the RK-A group regarding the tissue MPO levels Table 2 The median MPO levels for each study group. Group (n)
SHAM (n = 5) CONT-A (n = 6) MP-A (n = 7) RK-A (n = 7) SK-A (n = 7) CONT-SA (n = 6) MP-SA (n = 7) RK-SA (n = 7) SK-SA (n = 7)
Fig. 4. Tissue lipid peroxidation levels in the subacute stage groups. CONTSA = control; MP-SA = methylprednisolone; RK-SA = racemic ketamine; SKSA = (S)-ketamine groups of subacute stage; a = significant difference from the CONT-SA group.
MPO level Minimum
Maximum
Median
0.005 0.149 0.046 0.011 0.071 0.132 0.051 0.053 0.110
0.037 0.169 0.166 0.207 0.333 0.147 0.318 0.157 0.579
0.010* 0.158* 0.053* 0.037* 0.231* 0.135*,z 0.213*,z 0.099*,z 0.140*,z
n, number of rats; MPO, myeloperoxidation; CONT-A = control; MP-A = methylprednisolone; RK-A = racemic ketamine; SK-A = (S)-ketamine groups of acute stage; CONT-SA = control; MP-SA = methylprednisolone; RK-SA = racemic ketamine; SKSA = (S)-ketamine groups of subacute stage. * p = 0.001 among all groups. z p = 0.046 among subacute groups.
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(Fig. 5). Additionally, the MPO levels of the SHAM group were also statistically different from the subacute stage groups (Fig. 5). So, it can be said that except the racemic ketamine, none of the study drugs could block the increase of the myeloperoxidase enzyme originating from the lysosomes of the inflammatory cells neither in acute stage nor in subacute stage of SCI in rat. And their beneficial effects in SCI should be explained mainly by the other neuroprotective mechanisms such as decrease in mitochondrial dysfunctions, LPO, intracellular calcium overload. Study limitations This prospective experimental study has some pitfalls. Firstly, because there is no way knowing at which doses racemic ketamine should have been compared with (S)-ketamine to guarantee for ‘equi-neuroprotective’ drug efficacy at spinal cord level, we used equal doses of racemic ketamine and of (S)-ketamine intraperitonally to evaluate and to compare the possible neuroprotective effects of this agent and its enantiomers. Secondly, the study was designed as a preliminary study and because of the presence of the high risk of the urinary sepsis due to the SCI and death of the experimental animals, the study did not contain observations of functional outcome data and histopathological and biochemical evaluations occurring in the long run. Thirdly, this study could not be supported by immunohistochemical, and electron microscopic findings which can show ultrastructural details of the inflammatory response, neuronal necrosis and oedema in the acute and/or subacute stages of the SCI. Because the spinal cord of rat is too small to perform a large variety of the biochemical tests, this study could also not be supported by using more specific biochemical analyses for other detailed inflammatory pathways of the SCI (such as apoptotic pathways, glutathione level, nitrite/nitrate level, and xanthine oxidase activity level measurements). Additionally, we could not demonstrate the impact of the inflammatory reaction statistically because of some technical problems which blocked us to obtain and compare the inflammatory cell count results of the groups. And lastly, the combined effects of methylprednisolone and racemic ketamine or (S)-ketamine was not investigated in this study. Conclusions We have three major observations in this experimental study: (1) Both racemic ketamine and (S)-ketamine reduce LPO levels in the acute stage of SCI in rat. But only the effect of (S)-ketamine prolongs to the subacute stage. (2) Only racemic ketamine reduces MPO levels originating from the lysosomes of inflammatory cells in the acute stage of SCI in rat. But this protective effect does not prolong to the subacute stage. (3) Because, there was not a statistically significant difference among the study groups, it can be said that (S)-ketamine and racemic ketamine are as effective as methylprednisolone in preventing neuronal damage both in the acute stage and in the subacute stage of SCI in rat histopathologically.
In conclusion, (S)-ketamine and racemic ketamine are as effective as methylprednisolone in reducing the LPO levels in the acute stage of SCI and in preventing from secondary SCI histopathologically in both the acute and the subacute stages. In addition, (S)-ketamine is more effective than methylprednisolone in reducing the LPO levels in the subacute stage of SCI in rats. So, it can be suggested that the combined use of methylprednisolone with (S)-ketamine may provide an additional protective effect in SCI through different effect mechanisms.
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Conflict of interest statement The authors declare that they have no conflict of interest. References 1. Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 1991;75:15–26. 2. Kaynar MY, Hanci M, Kafadar A, Gu¨mu¨s¸tas¸ K, Belce A, Ciplak N. The effect of duration of compression on lipid peroxidation after experimental spinal cord injury. Neurosurg Rev 1998;21:117–20. 3. Aslan A, Cemek M, Eser O, Altunbas¸ K, Buyukokuroglu ME, Cosar M, et al. Does dexmedetomidine reduce secondary damage after spinal cord injury? An experimental study. Eur Spine J 2009;8:336–44. 4. Kaptanoglu E, Tuncel M, Palaoglu S, Konan A, Demirpenc¸e E, Kilinc¸ K. Comparison of the effects of melatonin and methylprednisolone in experimental spinal cord injury. J Neurosurg 2009;93:77–84. 5. Gu¨l S, Celik SE, Kalayci M, Tas¸yu¨rekli M, Cokar N, Bilge T. Dose-dependent neuroprotective effects of melatonin on experimental spinal cord injury in rats. Surg Neurol 2005;64:355–61. 6. Hoffman WE, Kochs E, Werner C, Thomas C, Albrecht RF. Dexmedetomidine improves neurologic outcome from incomplete ischemia in rat. Anesthesiology 1991;75:328–32. 7. Gaviria M, Privat A, d’Arbigny P. Neuroprotective effects of novel NMDA antagonist Gacylidine, after experimental contusive spinal cord injury in adult rats. Brain Res 2000;874:200–9. 8. Cosar M, Eser O, Fidan H, Sahin O, Buyukbas S, Ela Y, et al. The neuroprotective effect of dexmedetomidine in hippocampus of rabbits after subarachnoid hemorrhage. Surg Neurol 2009;71:54–9. 9. Merola A, O’Brian MF, Castro BA, Smith DA, Eule JM, Lowe TG, et al. Histologic characterization of acute spinal cord injury treated with intravenous methylprednisolone. Orthop Trauma 2002;16:155–61. 10. Taoka J, Okajima K, Uchiba M, Johno M. Methylprednisolone reduces spinal cord injury in rats without affecting tumor necrosis factor-alpha production. J Neurotrauma 2001;18:533–43. 11. Fu ES, Saporta S. Methylprednisolone inhibits production of interleukin-1beta and interleukin-6 in the spinal cord following compression injury in rats. J Neurosurg Anesthesiol 2005;17:82–5. 12. Bracken MB, Shepard MJ, Hellenbrand KG, Collins WF, Leo LS, Freeman DF, et al. Methylprednisolone or naloxone treatment after acute spinal cord injury: 1year follow-up data. Results of the Second National Acute Spinal Cord Injury Study. J Neurosurg 1992;76:23–31. 13. Bracken MB, Shepard MJ, Horford TR, Leo-Summers L, Aldrich EF, Fazl M, et al. Methylprednisolone or tirilazad mesylate administration after acute spinal cord injury: 1-year follow-up data. Results of the Third National Acute Spinal Cord Injury randomized controlled trial. J Neurosurg 1998;89:699–706. 14. Nesathurai S. Steroids and spinal cord injury: revisiting the NASCIS 2 and NASCIS 3 trials. J Trauma 1998;45:1088–93. 15. Sayer FT, Kronvall E, Nilsson OG. Methylprednisolone treatment in acute spinal cord injury: the myth challenged through a structured analysis of published literature. Spine J 2006;6:335–43. 16. Reich DL, Silvay G. Ketamine: an update on the first twenty-five years of clinical experience. Can J Anaesth 1989;36:186–97. 17. Himmelseher S, Pfenninger E, Georgieff M. The effects of ketamine-isomers on neuronal injury and regeneration in rat hippocampal neurons. Anesth Analg 1996;83:505–12. 18. Himmelseher S, Pfenninger E, Kochs E, Auchter M. S (+)-ketamine up-regulates neuronal regeneration associated proteins following glutamate injury in cultured rat hippocampal neurons. J Neurosurg Anesthesiol 2000;12:84–94. 19. Gonzales JM, Loeb AL, Reichard PS, Irvine S. Ketamine inhibits glutamate-Nmethyl-D-aspartate-, and quisqualate-stimulated cGMP production in cultured cerebral neurons. Anesthesiology 1995;82:205–13. 20. Orser BA, Pfenninger E, MacDonald JF. Multiple mechanisms of ketamine blockade of N-methyl-D-aspartate receptors. Anesthesiology 1997;86:903–17. 21. Proescholdt M, Heimann A, Kempski O. Neuroprotection of S() ketamine isomer in global forebrain ischemia. Brain Res 2001;904:245–51. 22. Engelhard K, Werner C, Eberspacher E, Bachl M, Blobner M, Hildt E, et al. The effect of alpha-2 agonist dexmedetomidine and N-methyl-D-aspartate antagonist S() ketamine on the expression of apopitosis-regulating proteins after incomplete cerebral ischemia and reperfusion in rats. Anesth Analg 2003;96:524–31. 23. Kawasaki T, Ogata M, Kawasaki C, Ogata J, Inoue Y, Shigematsu A. Ketamine suppresses proinflammatory cytokine production in human blood in vitro. Anesth Analg 1999;89:665–9. 24. Hoffman WE, Pelligrino D, Werner C, Kochs E, Albrecht RF, Schulte Am Esch J. Ketamine decreases plasma catecholamines and improves outcome from incomplete cerebral ischemia in rats. Anesthesiology 1992;76:755–62. 25. Kato R, Foex P. Myocardial protection by anesthetic agents against ischemia– reperfusion injury: an update for anesthesiologists. Can J Anaesth 2002;49: 777–91. 26. Salman AE, Dal D, Salman MA, Iskit AB, Aypar U. The effect of ketamine on acute muscular ischemia–reperfusion in rats. Eur J Anaesthesiol 2005;22:712–6. 27. Camara-Lemarroy CR, Guzman-de la Garza FJ, Alarcon-Galvan G, Cordero-Perez P, Fernandez-Garza NE. The effects of NMDA receptor antagonists over intestinal ischemia/reperfusion injury in rats. Eur J Pharmacol 2009;621:78–85.
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28. Lee HT, Ota-Setlik A, Fu Y, Nasr SH, Emala CW. Differential protective effects of volatile anesthetics against renal ischemia–reperfusion injury in vivo. Anesthesiology 2004;101:1313–24. 29. Rivlin AS, Tator CH. Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 1978;10:38–43. 30. Church J, Zeman S, Lodge D. The neuroprptective action of ketamine and MK801 after transient cerebral ischemia in rats. Anesthesiology 1998;69:702–9. 31. Black P, Markowitz RS, Cooper V, Mechanic A, Kushner H, Damjanov I, et al. Models of spinal cord injury: part 1. Static load technique. Neurosurgery 1986;19:752–62. 32. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1977;86:271–8. 33. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8.
34. Demirpenc¸e E, Ko¨ksoy C, Kuzu A, Kılınc¸ K. A spectrophotometric assay for tissue-associated myeloperoxidase activity and its application to intestinal ischemia–reperfusion. Turk J Med Sci 1997;27:197–200. 35. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assay method for myeloperoxidase in human polymorphonuclear leucocytes. Anal Biochem 1982;127: 346–50. 36. Torres S, Salgado-Ceballos H, Torres JL, Orozco-Suarez S, Dı´az-Ruı´z A, Martı´nez A, et al. Early metabolic reactivation versus antioxidant therapy after a traumatic spinal cord injury in adult rats. Neuropathology 2010;30: 36–43. 37. Christie SD, Comeau B, Myers T, Sadi D, Purdy M, Mendez I. Duration of lipid peroxidation after acute spinal cord injury in rats and the effect of methylprednisolone. Neurosurg Focus 2008;25(5):E5.
Contents lists available at SciVerse ScienceDirect
Injury journal homepage: www.elsevier.com/locate/injury
Neuroprotective effects of racemic ketamine and (S)-ketamine on spinal cord injury in rat Emine Arzu Kose a,*, Bulent Bakar b, Sebnem Kupana Ayva c, Kamer Kilinc d, Alpaslan Apan a a
Kirikkale University, School of Medicine, Department of Anaestesiology and Reanimation, Kirikkale, Turkey Kirikkale University, School of Medicine, Department of Neurosurgery, Kirikkale, Turkey c Kirikkale University, School of Medicine, Department of Pathology, Kirikkale, Turkey d Hacettepe University, School of Medicine, Department of Biochemistry, Ankara, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 29 February 2012
Background: The aim of this study was to investigate and to compare the potential neuroprotective effects of racemic ketamine, (S)-ketamine and methylprednisolone after an experimental spinal cord injury model in rats. Methods: Fifty-nine Wistar albino rats were divided into three main groups as acute stage (A), subacute stage (SA) and sham groups and then acute and subacute stage groups were divided into four groups regarding the used drug as control (CONT), racemic ketamine (RK), (S)-ketamine (SK) and methylprednisolone (MP) groups. A dorsal laminectomy was performed; and spinal cord injury was induced by using a temporary aneurysm clip. Four hours later from the clip compression, except those of the sham and control groups, the drugs (60 mg/kg racemic ketamine, 60 mg/kg (S)-ketamine or 30 mg/kg methylprednisolone) were administered intraperitoneally. At 72th h and 7th days of the study, the spinal cords of rats were removed from T8 level to the conus medullaris level. The specimens were and evaluated histopathologically, tissue lipid peroxidation (LPO) and myeloperoxidation (MPO) levels were measured and biochemically. Results: The histopathological results were similar both in the acute and in the subacute stage groups. There was a statistically significant difference among all groups regarding the tissue LPO levels (p < 0.001). There was a statistically significant difference between the CONT-A group and the MP-A, RKA and SK-A groups (p = 0.004, p < 0.001 and p = 0.007, respectively) in acute stage and between the CONT-SA group and SK-SA group (p = 0.002) in subacute stage. There was a statistically significant difference among all groups regarding the tissue MPO levels (p = 0.001). The median MPO levels were similar among acute stage groups (p = 0.057), but there was a statistical difference among subacute stage groups (p = 0.046). Conclusion: (S)-ketamine is more effective than methylprednisolone and racemic ketamine to reduce the LPO levels in subacute stage of spinal cord injury in rats. And, it is as effective as methylprednisolone in preventing secondary spinal cord injury histopathologically. ß 2012 Elsevier Ltd. All rights reserved.
Keywords: Racemic ketamine (S)-ketamine Methylprednisolone Spinal cord injury Rat
Introduction Traumatic spinal cord injury (SCI) is still a major clinical problem with permanent neurological deficits and secondary complications. The initial mechanical damage causing immediate cell death in the spinal cord is known as primary injury and inevitable. After primary injury, lesions greatly enlarge and worsen by secondary injury.1 Secondary injury mechanisms involve
* Corresponding author at: Kirikkale University, School of Medicine, Department of Anaestesiology and Reanimation, 71100-Kirikkale, Turkey. Tel.: +90 318 225 24 85/22 63; fax: +90 318 225 28 19. E-mail address: [email protected] (E.A. Kose). 0020–1383/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2012.02.022
excessive release of glutamate and aspartate, intracellular calcium overload, the activation of arachidonic acid cascade, and the induction of free radical induced lipid peroxidation (LPO) which has often been suggested to be an important factor in posttraumatic neuronal degeneration.2 Although the exact mechanism is unknown, altered blood flow and changes in microvascular permeability, as well as sympathetic stimuli including norepinephrine may contribute to the development of secondary injury.3–8 Methylprednisolone, a potent immunosupressive glucocorticoid, has beneficial effects in improving neurologic recovery when administered within 8 h after the onset of the SCI. It reduces the progression of oedema and has antioxidant and cell membrane stabilising properties.9 Taoka et al. showed that methylprednisolone
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reduces the severity of SCI by inhibiting activated leucocytes.10 The neuroprotective effect of methylprednisolone can be related to an interaction with the cytokine cascade and decrease in the production TNF-a and IL-6.11 Although the precise mechanism of action of methylprednisolone is not completely understood, it is the most commonly used agent as a standard care for SCI.12,13 While it is effective in experimental and clinical studies, the use of high-dose corticosteroids has recently been questioned.14,15 Therefore, an increasing number of studies now focus on potential neuroprotective agents against secondary injury.3–8 Ketamine is an anaesthetic agent, which inhibits N-methyl-Daspartate (NMDA) receptors. Ketamine has two enantiomers because of its molecular structure: S(+) ketamine and R() ketamine.16 Several experimental studies suggest that ketamine may be capable of producing neuroprotective effects.17–21 Racemic ketamine inhibits nitric oxide synthesis and nitric oxide-dependent cyclic guanosine monophosphate production stimulated by glutamate and glutamate analogues in primary culturs of cortical neurons and glia.19 This ketamine-induced inhibition of glutamate receptors may contribute to cellular protection against ischemic injury. The data obtained from experimental studies emphasize that the salutary actions of ketamine in models of neuronal injury appear to be stereoselective.17,18,21,22 While racemic and (S)- but not (R)-ketamine attenuate injury after glutamate exposure or axonal transection in rat hippocampal neurons in vitro, neuroregenerative effects appear only with (S)-ketamine.17 Ketamine may also decrease the severity of neuronal damage by interfering with the inflammatory response to ischemia. Ketamine suppresses lipopolysaccharide-induced TNF-a, IL-6, and IL-8 production and inhibits neutrophil adhesion to the endothelium in vitro.23 Ketamine also enhances neurologic outcome concomitant with reduction in plasma catecholamine concentrations in a rat model of incomplete cerebral ischemia.24 Additionally, it has been shown that, ketamine protects various tissues from ischemia/reperfusion (I/R) injury, such as myocardium, skeletal muscle, intestinal tissue, kidney and reduces malondialdehyde levels, a specific marker of LPO, in these tissues.25–28 To the best of our knowledge, there has been no research on the neuroprotective effects of ketamine-enantiomers after SCI. This study was designed to investigate and to compare the possible neuroprotective effects of racemic ketamine, (S)-ketamine and methylprednisolone after an experimental SCI model in rats.
Materials and methods Materials The investigation was conducted in accordance with the Guide for Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and approval has been received from the Animal Ethics Committee of The Ministry of Health Ankara Research and Training Hospital. Racemic ketamine (Ketalar1, Pfizer Inc., USA), (S)-ketamine (Ketanest1 S, Pfizer Inc., USA), and methylprednisolone (Depomedrol1, Pharmacia & Upjohn Company, Kalamazoo, USA) were used in this study. Fifty-nine male, Wistar albino rats weighing 250–300 g firstly were divided into three main groups as acute stage (A), subacute stage (SA) and sham groups; and then except the sham group which was not performed spinal cord injury, each main group was divided into four groups according to used study drug using a random numbers table. The acute stage study groups were named as following: - SHAM group (no SCI + no drug; n = 5).
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- CONT-A group (SCI + 1 mL % 0.9 NaCl intraperitoneal (i.p.); n = 6). - MP-A group (SCI + 30 mg/kg methylprednisolone in 1 mL volume i.p., followed by a maintenance dose of 5.4 mg/kg/h; n = 7). - RK-A group (SCI + 60 mg/kg racemic ketamine in 1 mL volume i.p.; n = 7). - SK-A group (SCI + 60 mg/kg (S)-ketamine in 1 mL volume i.p.; n = 7). The acute stage study groups were named as following: - CONT-SA group (SCI + 1 mL % 0.9 NaCl intraperitoneally i.p.; n = 6). - MP-SA group (SCI + 30 mg/kg methylprednisolone in 1 mL volume i.p., followed by a maintenance dose of 5.4 mg/kg/h; n = 7). - RK-SA group (SCI + 60 mg/kg racemic ketamine in 1 mL volume i.p.; n = 7). - SK-SA group (SCI + 60 mg/kg (S)-ketamine in 1 mL volume i.p.; n = 7). Methods All rats were anaesthetized via intramuscular injection of 30 mg/kg thiopental sodium (Penthal Sodyum, IE Ulugay Ilac San., I˙stanbul, Turkey) and 5 mg/kg xylazine HCl (Rompun1 %2, Bayer HealthCare AG, Germany); breathing was continued spontaneously with room air. Rats were positioned prone on operating table and a midline dorsal incision was done under a sterile technique. A dorsal laminectomy at thoracal 9–10 level was performed, and the dura mater was left intact. The spinal cord was exposed; and except SHAM group spinal cord injury was induced by using a temporary aneurysm clip (Mizuho1 Aneurysm Clip, Mizuho, Japan) by using a technique described before by Rivlin and Tator.29 The clip was removed after 60 s and the development of haemorrhagic contusion was seen at the point on the spinal cord where the clip was placed (Fig. 1) and then paravertebral fascia and skin were sutured with silk stitches. Four hours later from the clip compression, the study drugs were administered to rats via intraperitoneal injection using a 22G needle according to their included groups. In this study, methylprednisolone was administered in the doses of used in standard care protocol of SCI.12,13 As a maintenance dose, MP-A and MP-SA groups were given a total of 5.4 mg/kg/h intraperitoneal methylprednisolone treatment at 6 h intervals, with the total dose being 23 h. Additionally, due to the fact that Church et al. have shown that 60 mg/kg ketamine has neuroprotective effects in transient cerebral ischemia in rats, 60 mg/kg dose of ketamine was preferred in this study.30 After the administration of the study drugs, all rats were recovered from anaesthesia spontaneously under the blanket. Hind limb locomotor deficit resulted from SCI was observed in all rats which were clip compression was used. Seventy two hours later, all animals selected for acute stage groups; and 7 days later the animals selected for subacute stage groups were re-anaesthetized with intramuscular thiopenthal sodium and xylazine HCl and cardiac air embolization was performed for scarification. Dorsal incision of the animals was re-opened, and the spinal cord was totally removed from T8 level to the conus medullaris level. The spinal cord was divided into two parts horizontally. The proximal side included contusion lesion was stored in 10% buffered formaldehyde solution for histopathological examination at room temperature and the distal part was stored at 30 8C at dry air for biochemical examination. Specimen analysis For histopathological examination, all tissue samples were fixed at 10% buffered formaldehyde and processed according to
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ammonium bromide (HETAB) and 5 mM EDTA. The resulting suspension was centrifuged at 5000 g for 2 min and the supernatant was used for the activity measurement. The MPO activity was measured in a final volume of 1 mL containing 80 mM phosphate buffer (pH: 5.4), 0.5% HETAB, 1.6 mM synthetic substrate tetramethylbenzidine (TMB) initially dissolved in dimethylformamide, 2 mM H2O2 and the sample. The reaction was started at 37 8C by the addition of H2O2. The initial rate of MPO-catalyzed TMB oxidation was followed by recording the increase of absorbance at 655 nm (Shimadzu1 UV-120-02 spectrophotometry). The MPO activity was expressed as the amount of the enzyme producing one absorbance change per minute under assay conditions. Tissue-associated MPO activity was calculated as units per gram of wet tissue. Statistical analysis
Fig. 1. A dorsal laminectomy at thoracal 9–10 level was performed and traumatic spinal cord injury was induced by using a temporary aneurysm clip (a) exposed for 60 s (b).
Data were analysed using the SPSS 11.5 (SPSS Inc., Software, Chicago, Illinois, USA) statistical software. Tissue LPO levels were normally distributed and the variation was homogenous among all groups. Therefore, LPO levels were analyzed by using one-way analysis of variance (ANOVA) and with Tukey test so as to determine comparison among groups, and differences among groups, respectively. MPO levels were not normally distributed and the variation was not homogenous among all groups. So, MPO levels were analysed by using the Krusskal–Wallis test, and with the Mann–Whitney U test (with Bonferroni correction) so as to determine comparison among groups, and differences among groups, respectively. Histopathological data were analysed by using the Chi-Square test both to determine comparison among groups or differences among groups. p < 0.05 was considered statistically significant. Results
routine light microscopic tissue processing technique. Serial sections of 5 mm stained with haematoxylin–eosin were examined and photographed by a microscope (Leica1 Microsystems, Wetzlar GmbH). Each section was evaluated by experienced histopathologist blinded to the groups, and study drugs. For evaluation of the SCI histopathologically, a grading system described by Black et al. was used31 on all specimens as following: Grade 0: no destruction on the spinal cord tissue histopathologically. Grade I: mild neural tissue destruction and polymorphonuclear cell infiltration without neuronal cell lost; the posterior column of the spinal cord was affected Grade II: moderate neural tissue destruction and macrophage and/or histiocyte infiltration with white matter loss and central cavitation Grade III: severe neural tissue destruction with white and grey matter cystic necrosis and gliosis. Biochemical determinations were carried out by biochemist blinded to the animal groups, and test materials. Frozen tissue samples were weighted and homogenized in 1:10 (w:v) potassium phosphate buffer (50 mM, pH: 7.4) by using a dounce homogenizer. Thiobarbituric acid reactive substances (TBARS) were measured as an index of LPO by the method of Mihara et al.32,33 Tissue levels of lipid peroxides (as TBARS) were calculated as nanomole per gram wet tissue. Tissue-associated myeloperoxidation (MPO) activity was measured by the modified method of Suzuki et al.34,35 Tissue homogenate (0.5 mL) was centrifuged at 10,000 g for five minutes, and the pellet was resuspended in equal volume (0.5 mL) of 50 mM phosphate buffer (pH: 6.0) containing 0.5% hexadecyltrimethyl
Histopathological analysis Spinal cord sections from the sham group in 72 h after operation had normal histological structure (Fig. 2). There was a statistically significant difference among all groups regarding histopathological grades (X2 = 73.493, p < 0.001), and this significance was result from the SHAM group. There was a statistically significant difference neither among acute stage groups nor among the subacute stage groups regarding the histopathological grades (X2 = 6.396, p = 0.380; and X2 = 2.250, p = 0.522, respectively). The histopathological grades assigned for each group and the light microscopic appearance for each histopathological grade was shown in Table 1 and Fig. 2, respectively. Biochemical analysis There was a statistically significant difference among all groups regarding the tissue LPO levels (F = 6.591, p < 0.001), and this statistically significant difference was also observed either among acute stage groups (F = 12.150, p < 0.001) or among the subacute stage groups (F = 5.813, p = 0.004). There was a statistically significant difference between the CONT-A/MP-A, CONT-A/RK-A and CONT-A/SK-A groups (p = 0.004, p < 0.001 and p = 0.007, respectively) in acute stage (Fig. 3). When posthoc comparisons were made, no statistically difference was observed between the CONT-SA/MP-SA, and the CONT-SA/RK-SA groups (p = 0.062 and p = 0.061, respectively) but there was a statistically difference between the CONT-SA group and SK-SA group (p = 0.002) in subacute stage (Fig. 4). Mean LPO levels of the SHAM group were also statistically different from the CONT-A group (p < 0.001) and the CONT-SA group (p = 0.007) (Figs. 3 and 4).
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Fig. 2. Histopathological grades: Grade 0 = no neural tissue destruction. Grade I = mild neural tissue destruction and polymorphonuclear cell infiltration without neuronal cell lost; the posterior column of the spinal cord was affected. Grade II = moderate neural tissue destruction and macrophage and/or histiocyte infiltration with white matter lost and central cavitation. Grade III = severe neural tissue destruction with white and grey matter cystic necrosis and gliosis (H&E100, and H&E400).
There was a statistically significant difference among all groups regarding the tissue MPO levels (X2 = 27.259, p = 0.001). Nevertheless, when paired comparisons were made, no statistical significance was determined between compared groups. The median MPO levels were not statistically different among acute stage groups (X2 = 7.539, p = 0.057), but there was a statistical difference among subacute stage groups (X2 = 7.981, p = 0.046). However, no statistical significant difference was determined by paired comparisons. The median MPO levels for each group were shown in Table 2. Additionally, there was a statistically significant difference between the SHAM group and each of the other study groups except the RK-A group (p = 0.050) regarding the tissue MPO levels, when paired comparisons were made and Bonferroni correction was performed (Fig. 5). Discussion
Additionally, these results were in favour of (S)-ketamine in preventing neuronal damage in acute stage of SCI. When subacute stage groups were investigated, moderate to severe inflammatory reaction containing macrophages and histiocytes was observed. So, it can be said that none of the study drugs could reduce or completely block the macrophage and histiocyte infiltration into the injured neuronal tissue in the subacute stage of SCI. Although, there was not a statistical significance among subacute stage groups, the frequency of the Grade III degeneration was the lowest with a 14.3% frequency in SK-SA group; and it was higher in the MP-SA group than RK-SA group with 42.9% and 28.6% frequencies, respectively. On the base of these results, it can be suggested that, (S)-ketamine has a trend towards to be more effective agent than both methyprednisolone and racemic ketamine in preventing neuronal damage in subacute stage of SCI in rat as well as in acute stage and the beneficial effects of ketamine are stereoselective.17–21
Histopathological evaluation Biochemical evaluation In this study, there was mild to severe degree of neuronal destruction with demyelination and cavity formation in all acute stage groups except the SHAM group (Fig. 2). Grade III degeneration was lowest with a 14.3% frequency in SK-A group (Table 1). Because, there was not a statistical significance among acute stage groups regarding the histopathological grades, it can be said that racemic ketamine and (S)-ketamine are as effective as methyprednisolone to reduce the severity of neuronal damage by interfering with the inflammatory response to ischemia.11,23
Lipid peroxidation initiated by oxidative stress is one of the most important and destroying effects of ‘‘the free radicals following SCI. SCI disturbs the ability of mitochondria to carry out cellular respiration and oxidative phosphorylation. Ischemia also challenges tissue energy demands and function of active ion channels and cells then switch from aerobic to anaerobic metabolism and lead to cell death.36 When the LPO levels were evaluated in acute stage groups it was seen that there was a
Table 1 The histopathological grades for each group. Group Acute stage
Grade
Grade Grade Grade Grade
0 I II III
Subacute stage
SHAM
CONT
MP
RK
SK
CONT
MP
RK
SK
5(100) 0(0) 0(0) 0(0)
0(0) 1(16.7) 2(33.3) 3(50.0)
0(0) 0(0) 4(57.1) 3(42.9)
0(0) 0(0) 4(57.1) 3(42.9)
0(0) 0(0) 6(85.7) 1(14.3)
0(0) 0(0) 3(50.0) 3(50.0)
0(0) 0(0) 4(57.1) 3(42.9)
0(0) 0(0) 5(71.4) 2(28.6)
0(0) 0(0) 6(85.7) 1(14.3)
Data are expressed as numbers, with percentiles in parenthesis. CONT = control; MP = methylprednisolone; RK = racemic ketamine; SK = (S)-ketamine.
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Fig. 3. Tissue lipid peroxidation levels in the acute stage groups. CONT-A = control; MP-A = methylprednisolone; RK-A = racemic ketamine; SK-A = (S)-ketamine groups of acute stage; a = significant difference from the CONT-A group; b = significant difference from the SHAM group.
Fig. 5. Tissue myeloperoxidation levels in the acute and subacute stage groups. CONT-A = control; MP-A = methylprednisolone; RK-A = racemic ketamine; SKA = (S)-ketamine groups of acute stage; CONT-SA = control; MPSA = methylprednisolone; RK-SA = racemic ketamine; SK-SA = (S)-ketamine groups of subacute stage; a = significant difference from the SHAM group.
statistically significant difference between the CONT-A/MP-A, CONT-A/RK-A, and CONT-A/SK-A groups; but there was no statistical difference between the SHAM group and the study groups which were study drugs was given (Fig. 3). So, it can be said that all of the study drugs could reduce the LPO levels and both racemic ketamine and (S)-ketamine are as effective as methylprednisolone in acute stage of SCI in rat. This beneficial effect of ketamine can be explained by different mechanisms. Ketamine, a NMDA receptor antagonist, blockades an ion channel that is permeable to Ca++ and glutamate release by ischemic neurons. The activation of these receptors increases Ca++ influx and rapidly initiates cell necrosis and apoptosis. Thus, the blockade of NMDA receptors may potentially inhibits this excitotoxic injury.17,18 It has been shown that ketamine inhibits nitric oxide synthesis and nitric oxide-dependent cyclic guanosine monophosphate production stimulated by glutamate and glutamate analogues (e.g., NMDA, quisqualate, kainite) in primary culturs of cortical neurons and glia.19 This ketamine-induced inhibition of glutamate receptors
may contribute cellular protection against ischemic injury. Ketamine may also decrease the severity of neuronal damage by interfering with the inflammatory response to ischemia by suppressing lipopolysaccharide-induced TNF-a, IL-6, and IL-8 production and inhibiting neutrophil adhesion to the endothelium.23 When the LPO levels in subacute stage groups were evaluated, it was seen that the only statistical difference was between the CONT-SA group and SK-SA group, and there was no statistical difference between the SHAM group and the SK-SA group (Fig. 4). Thus, it can be said that among the study drugs only (S)-ketamine has a beneficial effect which prolongs to subacute stage. Neutrophils and other phagocytes produce hypochloride, a strong oxidant when the enzyme myeloperoxidase acts on hydrogen peroxide and chloride ion. Myeloperoxidase is a specific enzyme in large quantities in granules of the neutrophils and MPO activity is correlated with the absolute number of the neutrophils and their activations. Neutrophils infiltration into the injured spinal cord tissue occurs in 6 h and is followed by activated macrophages and microglia. It peaks in 24–48 h and may persist during the first week.37 In this study, there was a statistically significant difference between the SHAM group and the acute stage groups except the RK-A group regarding the tissue MPO levels Table 2 The median MPO levels for each study group. Group (n)
SHAM (n = 5) CONT-A (n = 6) MP-A (n = 7) RK-A (n = 7) SK-A (n = 7) CONT-SA (n = 6) MP-SA (n = 7) RK-SA (n = 7) SK-SA (n = 7)
Fig. 4. Tissue lipid peroxidation levels in the subacute stage groups. CONTSA = control; MP-SA = methylprednisolone; RK-SA = racemic ketamine; SKSA = (S)-ketamine groups of subacute stage; a = significant difference from the CONT-SA group.
MPO level Minimum
Maximum
Median
0.005 0.149 0.046 0.011 0.071 0.132 0.051 0.053 0.110
0.037 0.169 0.166 0.207 0.333 0.147 0.318 0.157 0.579
0.010* 0.158* 0.053* 0.037* 0.231* 0.135*,z 0.213*,z 0.099*,z 0.140*,z
n, number of rats; MPO, myeloperoxidation; CONT-A = control; MP-A = methylprednisolone; RK-A = racemic ketamine; SK-A = (S)-ketamine groups of acute stage; CONT-SA = control; MP-SA = methylprednisolone; RK-SA = racemic ketamine; SKSA = (S)-ketamine groups of subacute stage. * p = 0.001 among all groups. z p = 0.046 among subacute groups.
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(Fig. 5). Additionally, the MPO levels of the SHAM group were also statistically different from the subacute stage groups (Fig. 5). So, it can be said that except the racemic ketamine, none of the study drugs could block the increase of the myeloperoxidase enzyme originating from the lysosomes of the inflammatory cells neither in acute stage nor in subacute stage of SCI in rat. And their beneficial effects in SCI should be explained mainly by the other neuroprotective mechanisms such as decrease in mitochondrial dysfunctions, LPO, intracellular calcium overload. Study limitations This prospective experimental study has some pitfalls. Firstly, because there is no way knowing at which doses racemic ketamine should have been compared with (S)-ketamine to guarantee for ‘equi-neuroprotective’ drug efficacy at spinal cord level, we used equal doses of racemic ketamine and of (S)-ketamine intraperitonally to evaluate and to compare the possible neuroprotective effects of this agent and its enantiomers. Secondly, the study was designed as a preliminary study and because of the presence of the high risk of the urinary sepsis due to the SCI and death of the experimental animals, the study did not contain observations of functional outcome data and histopathological and biochemical evaluations occurring in the long run. Thirdly, this study could not be supported by immunohistochemical, and electron microscopic findings which can show ultrastructural details of the inflammatory response, neuronal necrosis and oedema in the acute and/or subacute stages of the SCI. Because the spinal cord of rat is too small to perform a large variety of the biochemical tests, this study could also not be supported by using more specific biochemical analyses for other detailed inflammatory pathways of the SCI (such as apoptotic pathways, glutathione level, nitrite/nitrate level, and xanthine oxidase activity level measurements). Additionally, we could not demonstrate the impact of the inflammatory reaction statistically because of some technical problems which blocked us to obtain and compare the inflammatory cell count results of the groups. And lastly, the combined effects of methylprednisolone and racemic ketamine or (S)-ketamine was not investigated in this study. Conclusions We have three major observations in this experimental study: (1) Both racemic ketamine and (S)-ketamine reduce LPO levels in the acute stage of SCI in rat. But only the effect of (S)-ketamine prolongs to the subacute stage. (2) Only racemic ketamine reduces MPO levels originating from the lysosomes of inflammatory cells in the acute stage of SCI in rat. But this protective effect does not prolong to the subacute stage. (3) Because, there was not a statistically significant difference among the study groups, it can be said that (S)-ketamine and racemic ketamine are as effective as methylprednisolone in preventing neuronal damage both in the acute stage and in the subacute stage of SCI in rat histopathologically.
In conclusion, (S)-ketamine and racemic ketamine are as effective as methylprednisolone in reducing the LPO levels in the acute stage of SCI and in preventing from secondary SCI histopathologically in both the acute and the subacute stages. In addition, (S)-ketamine is more effective than methylprednisolone in reducing the LPO levels in the subacute stage of SCI in rats. So, it can be suggested that the combined use of methylprednisolone with (S)-ketamine may provide an additional protective effect in SCI through different effect mechanisms.
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Conflict of interest statement The authors declare that they have no conflict of interest. References 1. Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 1991;75:15–26. 2. Kaynar MY, Hanci M, Kafadar A, Gu¨mu¨s¸tas¸ K, Belce A, Ciplak N. The effect of duration of compression on lipid peroxidation after experimental spinal cord injury. Neurosurg Rev 1998;21:117–20. 3. Aslan A, Cemek M, Eser O, Altunbas¸ K, Buyukokuroglu ME, Cosar M, et al. Does dexmedetomidine reduce secondary damage after spinal cord injury? An experimental study. Eur Spine J 2009;8:336–44. 4. Kaptanoglu E, Tuncel M, Palaoglu S, Konan A, Demirpenc¸e E, Kilinc¸ K. Comparison of the effects of melatonin and methylprednisolone in experimental spinal cord injury. J Neurosurg 2009;93:77–84. 5. Gu¨l S, Celik SE, Kalayci M, Tas¸yu¨rekli M, Cokar N, Bilge T. Dose-dependent neuroprotective effects of melatonin on experimental spinal cord injury in rats. Surg Neurol 2005;64:355–61. 6. Hoffman WE, Kochs E, Werner C, Thomas C, Albrecht RF. Dexmedetomidine improves neurologic outcome from incomplete ischemia in rat. Anesthesiology 1991;75:328–32. 7. Gaviria M, Privat A, d’Arbigny P. Neuroprotective effects of novel NMDA antagonist Gacylidine, after experimental contusive spinal cord injury in adult rats. Brain Res 2000;874:200–9. 8. Cosar M, Eser O, Fidan H, Sahin O, Buyukbas S, Ela Y, et al. The neuroprotective effect of dexmedetomidine in hippocampus of rabbits after subarachnoid hemorrhage. Surg Neurol 2009;71:54–9. 9. Merola A, O’Brian MF, Castro BA, Smith DA, Eule JM, Lowe TG, et al. Histologic characterization of acute spinal cord injury treated with intravenous methylprednisolone. Orthop Trauma 2002;16:155–61. 10. Taoka J, Okajima K, Uchiba M, Johno M. Methylprednisolone reduces spinal cord injury in rats without affecting tumor necrosis factor-alpha production. J Neurotrauma 2001;18:533–43. 11. Fu ES, Saporta S. Methylprednisolone inhibits production of interleukin-1beta and interleukin-6 in the spinal cord following compression injury in rats. J Neurosurg Anesthesiol 2005;17:82–5. 12. Bracken MB, Shepard MJ, Hellenbrand KG, Collins WF, Leo LS, Freeman DF, et al. Methylprednisolone or naloxone treatment after acute spinal cord injury: 1year follow-up data. Results of the Second National Acute Spinal Cord Injury Study. J Neurosurg 1992;76:23–31. 13. Bracken MB, Shepard MJ, Horford TR, Leo-Summers L, Aldrich EF, Fazl M, et al. Methylprednisolone or tirilazad mesylate administration after acute spinal cord injury: 1-year follow-up data. Results of the Third National Acute Spinal Cord Injury randomized controlled trial. J Neurosurg 1998;89:699–706. 14. Nesathurai S. Steroids and spinal cord injury: revisiting the NASCIS 2 and NASCIS 3 trials. J Trauma 1998;45:1088–93. 15. Sayer FT, Kronvall E, Nilsson OG. Methylprednisolone treatment in acute spinal cord injury: the myth challenged through a structured analysis of published literature. Spine J 2006;6:335–43. 16. Reich DL, Silvay G. Ketamine: an update on the first twenty-five years of clinical experience. Can J Anaesth 1989;36:186–97. 17. Himmelseher S, Pfenninger E, Georgieff M. The effects of ketamine-isomers on neuronal injury and regeneration in rat hippocampal neurons. Anesth Analg 1996;83:505–12. 18. Himmelseher S, Pfenninger E, Kochs E, Auchter M. S (+)-ketamine up-regulates neuronal regeneration associated proteins following glutamate injury in cultured rat hippocampal neurons. J Neurosurg Anesthesiol 2000;12:84–94. 19. Gonzales JM, Loeb AL, Reichard PS, Irvine S. Ketamine inhibits glutamate-Nmethyl-D-aspartate-, and quisqualate-stimulated cGMP production in cultured cerebral neurons. Anesthesiology 1995;82:205–13. 20. Orser BA, Pfenninger E, MacDonald JF. Multiple mechanisms of ketamine blockade of N-methyl-D-aspartate receptors. Anesthesiology 1997;86:903–17. 21. Proescholdt M, Heimann A, Kempski O. Neuroprotection of S() ketamine isomer in global forebrain ischemia. Brain Res 2001;904:245–51. 22. Engelhard K, Werner C, Eberspacher E, Bachl M, Blobner M, Hildt E, et al. The effect of alpha-2 agonist dexmedetomidine and N-methyl-D-aspartate antagonist S() ketamine on the expression of apopitosis-regulating proteins after incomplete cerebral ischemia and reperfusion in rats. Anesth Analg 2003;96:524–31. 23. Kawasaki T, Ogata M, Kawasaki C, Ogata J, Inoue Y, Shigematsu A. Ketamine suppresses proinflammatory cytokine production in human blood in vitro. Anesth Analg 1999;89:665–9. 24. Hoffman WE, Pelligrino D, Werner C, Kochs E, Albrecht RF, Schulte Am Esch J. Ketamine decreases plasma catecholamines and improves outcome from incomplete cerebral ischemia in rats. Anesthesiology 1992;76:755–62. 25. Kato R, Foex P. Myocardial protection by anesthetic agents against ischemia– reperfusion injury: an update for anesthesiologists. Can J Anaesth 2002;49: 777–91. 26. Salman AE, Dal D, Salman MA, Iskit AB, Aypar U. The effect of ketamine on acute muscular ischemia–reperfusion in rats. Eur J Anaesthesiol 2005;22:712–6. 27. Camara-Lemarroy CR, Guzman-de la Garza FJ, Alarcon-Galvan G, Cordero-Perez P, Fernandez-Garza NE. The effects of NMDA receptor antagonists over intestinal ischemia/reperfusion injury in rats. Eur J Pharmacol 2009;621:78–85.
1130
E.A. Kose et al. / Injury, Int. J. Care Injured 43 (2012) 1124–1130
28. Lee HT, Ota-Setlik A, Fu Y, Nasr SH, Emala CW. Differential protective effects of volatile anesthetics against renal ischemia–reperfusion injury in vivo. Anesthesiology 2004;101:1313–24. 29. Rivlin AS, Tator CH. Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 1978;10:38–43. 30. Church J, Zeman S, Lodge D. The neuroprptective action of ketamine and MK801 after transient cerebral ischemia in rats. Anesthesiology 1998;69:702–9. 31. Black P, Markowitz RS, Cooper V, Mechanic A, Kushner H, Damjanov I, et al. Models of spinal cord injury: part 1. Static load technique. Neurosurgery 1986;19:752–62. 32. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1977;86:271–8. 33. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8.
34. Demirpenc¸e E, Ko¨ksoy C, Kuzu A, Kılınc¸ K. A spectrophotometric assay for tissue-associated myeloperoxidase activity and its application to intestinal ischemia–reperfusion. Turk J Med Sci 1997;27:197–200. 35. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assay method for myeloperoxidase in human polymorphonuclear leucocytes. Anal Biochem 1982;127: 346–50. 36. Torres S, Salgado-Ceballos H, Torres JL, Orozco-Suarez S, Dı´az-Ruı´z A, Martı´nez A, et al. Early metabolic reactivation versus antioxidant therapy after a traumatic spinal cord injury in adult rats. Neuropathology 2010;30: 36–43. 37. Christie SD, Comeau B, Myers T, Sadi D, Purdy M, Mendez I. Duration of lipid peroxidation after acute spinal cord injury in rats and the effect of methylprednisolone. Neurosurg Focus 2008;25(5):E5.