Curcumin improves early functional results after experimental spinal cord injury
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- Cemil, B., Topuz, K., Demircan, M.N. et al. Acta Neurochir (2010) 152: 1583. doi:10.1007/s00701-010-0702-x
Curcumin is a polyphenol extracted from the rhizome of Curcuma longa and well known as a multifunctional drug with anti-oxidative, anticancerous, and anti-inflammatory activities. The aim of the study was to evaluate and compare the effects of the use of the curcumin and the methylprednisolone sodium succinate (MPSS) functionally, biochemically, and pathologically after experimental spinal cord injury (SCI).
Forty rats were randomly allocated into five groups. Group 1 was performed only laminectomy. Group 2 was introduced 70-g closing force aneurysm clip injury. Group 3 was given 30 mg/kg MPSS intraperitoneally immediately after the trauma. Group 4 was given 200 mg/kg of curcumin immediately after the trauma. Group 5 was the vehicle, and immediately after trauma, 1 mL of rice bran oil was injected. The animals were examined by inclined plane score and Basso–Beattie–Bresnahan scale 24 h after the trauma. At the end of the experiment, spinal cord tissue samples were harvested to analyze tissue concentrations of malondialdehyde (MDA) levels, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity, and catalase (CAT) activity and pathological evaluation.
Curcumin treatment improved neurologic outcome, which was supported by decreased level of tissue MDA and increased levels of tissue GSH-Px, SOD, and CAT activity. Light microscopy results also showed preservation of tissue structure in the treatment group.
This study showed the neuroprotective effects of curcumin on experimental SCI model. By increasing tissue levels of GSH-Px, SOD, and CAT, curcumin seems to reduce the effects of injury to the spinal cord, which may be beneficial for neuronal survival.
KeywordsCurcuminMethylprednisoloneNeuroprotectionOxygen-free radicalSpinal cord injury
Following the trauma, the spinal cord undergoes an initial physical insult (primary injury) that is followed by a progressive injury process (secondary injury) and leads to tissue destruction . Primary injury is the initial mechanical damage to the spinal cord that results in tissue necrosis and loss of function and disruption of neural and vascular structures, whereas secondary injury is progressive cell injury that collectively damages intact, neighboring tissue [13, 30]. It has been suggested that one of the most important factors precipitating posttraumatic degeneration in the spinal cord is oxygen-free radical-induced lipid peroxidation (LP) [2, 17, 38]. Previously, much attention has been focused on the secondary injury because it appears to be vulnerable to pharmacological intervention [23, 25, 26, 43, 46]. There have been many studies investigating pharmacological agents such as methylprednisolone (MPSS), metoprolol, melatonin, erythropoietin, magnesium, infliximab, mexiletine, and naloxone, which protect or reduce secondary injury after experimental SCI [5, 23, 25–27, 43, 46].
Curcumin (diferuloylmethane) [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is a major yellow pigment in turmeric (the ground rhizome of Curcuma longa Linn), which is widely used as a spice and a coloring agent in several foods such as curry, mustard, and potato chips as well as in cosmetics and drugs [20, 33]. It shows a wide range of pharmacological activities, including antioxidant and anti-inflammatory effects . Curcumin has long been used in Asian medicine for its medical properties . It has been reported that administration of curcumin can ameliorate ischemia–reperfusion injury in myocardium , liver , kidney , brain , and other organ systems.
To date, the effect of the curcumin has not been evaluated biochemically after SCI. The aim of the present study was to investigate and compare the effects of curcumin and MPSS on the levels of malondialdehyde (MDA), GSH-Px, SOD, and CAT in tissue, on the light microscope, and on early functional results after experimental SCI in rats.
Methods and materials
Forty male Wistar rats (240–300 g), bred in the central animal house of Marmara University (Istanbul, Turkey), were used. The animals were housed under standard conditions of light and dark cycle with free access to food and tap water. All the protocols were approved by the institutional animal ethics committee of the Marmara University. The rats were randomly and blindly allocated into five groups, eight rats each. In group 1 (control), laminectomy was performed, and nontraumatized spinal cord samples were obtained 24 h after surgery. In group 2 (trauma), the animals underwent traumatic SCI after laminectomy. Injured spinal cord samples were removed 24 h after the trauma. In group 3 (MPSS), the animals received single doses of methylprednisolone sodium succinate (30 mg/kg; Mustafa Nevzat, Istanbul, Turkey) intraperitoneally, immediately after the trauma. In group 4 (curcumin), the animals received 200 mg/kg of curcumin (Sigma Chemicals Corp, St. Louis, MO, USA) intraperitoneally , immediately after the injury. In group 5 (vehicle), the animals received 1.0 mL of vehicle solution containing rice bran oil intraperitoneally, immediately after the injury .
Anesthesia and surgical procedure
The surgical procedure was performed with the animals under general anesthesia induced by intramuscular 50 mg/kg of ketamine hydrochloride (Ketalar, Pfizer, Istanbul, Turkey) and 10 mg/kg of xylazine (Rompun, Bayer, Istanbul, Turkey). The rats were placed in prone position. After T5 through T9 midline skin incision, the paravertebral muscles were dissected. T6 through T8 spinous processes were removed, and laminectomy was performed. The dura mater was left intact. Then, 70-g closing force aneurysm clip (Yasargil FE 721, Aesculap, Istanbul, Turkey) was applied onto the T7 level spinal cord for 1 min . The surgical wound was closed in layers with silk sutures after the procedure. Twenty-four hours after the trauma, anesthesia was reintroduced. Spinal cord samples (15 mm) were obtained from the operated spinal cord area and divided into two equal parts; then, the animals were sacrificed. Cranial parts of the tissue samples were obtained for microscopy evaluation; caudal parts were cleaned of blood with a scalpel and immediately stored in a −20°C freezer for biochemical analysis.
Measurement of MDA, GSH-Px, SOD, and CAT enzyme activities
The tissue samples were homogenized with 1 mL distilled water in an Ultra Turrax tissue homogenizer (T25 Janke & Kunkel GMBH, IKA-Labortechnik, Staufen, Germany) at 16,000 rpm for 5 min. The homogenates were used to measure the level of MDA, GSH-Px, SOD, and CAT activity. All the procedures were performed at 4°C. The protein content of the spinal cord was measured using the method of Lowry et al. .
LP in the spinal cord samples was determined as MDA concentration. The MDA levels were estimated by the NWLSS NWKMDA01 assay (Northwest Life Science Specialties), and activity was expressed as micromoles per gram protein. The GPX activity of the spinal cord was measured using a colorimetric assay kit (catalog number NWK-GPX01, NWLSS™ GPX assay, Northwest Life Science Specialties, Vancouver, WA, USA), which is based upon the method described by Paglia and Valentine . The GSH-Px concentration was calculated as milliunits per gram protein. SOD activity was determined using superoxide dismutase assay kit (catalog no.: 706002—Cayman Chemical, Ann Arbor, MI, USA). Activity was expressed as units per gram protein. CAT activity was measured using Catalase Assay Kit, Cat 707002 (Cayman Chemical, Ann Arbor, MI, USA). Activity was expressed as micromoles per gram protein.
Light microscopy evaluation
The specimen was immersed into 10% buffered formalin and stored at 4°C. One week after the sampling, they were removed from the store and placed in fresh fixative. Fixed tissue samples were processed routinely by paraffin-embedding technique. Sections of 6 µm were obtained on the coronal plane with microtome (Leica RM 2035, Germany; Leica RM2035, Leica Microsystem GmbH, Nussloch, Germany) and stained with hematoxylin–eosin. The preparations were evaluated using a light microscope (Olympus BH-2, Japan; Olympus BH-2, Olympus Corp, Tokyo, Japan) and were photographed (Sony CCD-IRIS, Sony, Inc., Tokyo, Japan). All the sections were evaluated morphologically by the same pathologist who was blinded to the treatment groups for presence of hemorrhage, cellular edema, and neutrophil infiltration.
Histopathological findings were graded as 1, 2, and 3 . Grade 1 indicates the appearance of a normal spinal cord. Grades 2 and 3 represent cellular edema with occasional hemorrhage and more severe edema with severe hemorrhage, respectively. The specimens of the groups were compared with sections of the control group not suffering from a traumatic episode.
Functional evaluation was applied to the rats 24 h after the trauma. The motor function of the rats was assessed in a blind manner by the inclined plane technique of Rivlin and Tator , and by the Basso, Beattie, and Bresnahan (BBB) scoring system described by Basso et al. . First, the technique of Rivlin and Tator was used to evaluate the maximum sloping, during which rats maintain themselves for 5 s, by the inclined plane (IP) test. The BBB scale ranges from 0 to 21. A score of “0” indicates that the animal exhibits complete hindlimb paralysis. A score of “21” denotes that the animal has completely normal locomotor function in the hindlimbs.
The Statistical Package for the Social Sciences version 11.0 (SPSS, Chicago, IL, USA) was used to perform statistical analyses. The results were expressed as mean ± SD. Statistical comparisons between the groups were tested with the Kruskal–Wallis test, and the Mann–Whitney U test was used for dual comparisons. In addition, pathological grading was statistically analyzed using a standard chi-square test. Values of P < 0.05 were considered statistically significant.
Activity of MDA
Activity of GSH-Px
Activity of SOD
Activity of CAT
Light microscopy findings
Results of pathologic examination
Control (n = 8)
Trauma (n = 8)
MPSS (n = 8)
Curcumin (n = 8)
Vehicle (n = 8)
IP scores of the control group and experimental groups
Mean ± SD
Control (n = 8)
84.37 ± 3.50**
Trauma (n = 8)
23.37 ± 10.57*
MPSS (n = 8)
50.87 ± 8.94*,**
Curcumin (n = 8)
53.12 ± 7.49*,**,****
Vehicle (n = 8)
29.62 ± 11.26***
BBB scores of the control group and experimental groups
Mean ± SD
Control (n = 8)
20.75 ± 0.71**
Trauma (n = 8)
2.75 ± 1.91*
MPSS (n = 8)
6.12 ± 3.94***
Curcumin (n = 8)
7.12 ± 3.68*,**,****
Vehicle (n = 8)
3.25 ± 1.91***
After a spinal cord trauma, the primary lesion is extended as a result of a series of autodestructive mechanisms, which conform to the secondary lesion. The latter originate a gradual degeneration of spinal cord parenchyma leading to chronic neurodegeneration . One of the major secondary mechanisms after traumatic injury is the free radicals' attack to the cellular membrane , a damaging mechanism altering all the cellular components including unsaturated fatty acids by the process of LP . Frequently used biomarker providing an indication of the overall LP level is the plasma concentration of MDA . In our study, high MDA levels were determined in the trauma group in comparison with the control group.
Metabolic bursts, in which oxygen is reduced to superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (OH−), can be elicited by various stimuli [11, 12]. SOD eliminates superoxides by converting them to hydrogen peroxide (H2O2). H2O2 is reduced to water by cytosolic antioxidants, CAT, and GSH-Px . The central nervous system is especially sensitive to damage associated with free radicals due to its high metabolic rate, high levels of polyunsaturated lipids, and its relatively low level of protective systems . In the present study, we have demonstrated that tissue levels of SOD, GSH-Px, and CAT are significantly decreased in the trauma group when compared to the control group.
MPSS is well accepted as an antioxidant anti-inflammatory agent that limits secondary injury after SCI [21, 27]. It was found that the ability of MPSS to improve blood flow and thus reduce the accumulation of lactate in the injured cord requires high tissue levels of glucocorticoid . Furthermore, MPSS inhibits posttraumatic LP and free radical generation and improves neurological function after traumatic SCI [7, 37]. Previously, Kalayci et al. and Yang and Piao reported that SCI significantly increases spinal cord tissue MDA levels and decreases SOD and GSH-Px enzyme activities. In addition, they showed that MPSS treatment decreases tissue MDA levels and prevents inhibition of the enzymes SOD and GSH-Px in the tissues [22, 48]. In the present study, increased tissue levels of GSH-Px, SOD, and CAT and decreased tissue levels of MDA were found in the MPSS group. These MPSS results were compatible with the findings in the studies by Kalayci et al. and Yang and Piao.
Curcumin has been found to possess a variety of pharmacological and biological activities , and a particular interest in this substance, in view of a potential clinical use, comes from the fact that it is a nontoxic and nonmutagenic natural product . Free radical scavenging activity of curcumin and its protective effect against reactive species are well documented . Curcumin is unique compared to other natural antioxidants since it possesses both the phenolic and diketonic groups which help in the scavenging of free radicals. In contrast, other natural antioxidants possess either phenolic or diketonic groups . One of the characteristic properties of curcumin is that it does not affect the normal cells . In a study on gerbils, it was reported that polyphenol-enriched curcumin might cross the blood–brain barrier . Sreejayan and Rao claimed that the curcumin inhibited iron-catalyzed LP in rat brain tissue homogenates by chelation of iron . Recently in the literature, Zhao et al. reported that curcumin inhibits oxidative stress and improves outcomes after focal cerebral ischemia by decreasing neuronal apoptosis . Following, Lin et al. reported that curcumin inhibited apoptosis and neuron loss, quenched astrocyte activation, and significantly improved BBB scores 7 day after spinal cord hemisection by downregulating GFAP expression . In the present study, the tissue levels of GSH-Px, SOD, and CAT were significantly increased in the curcumin group. In addition, it has also been shown that curcumin decreases the MDA level in the spinal cord. When the pathological sections were analyzed, the sections of the curcumin group demonstrated marked reduction of the histological features of SCI, consisting of more focal and mild hemorrhage, edema, and neutrophil infiltration. Furthermore, the curcumin group showed significantly improved pathological grades than the other experimental groups. The IP scores and BBB scores of the curcumin group were better than the trauma group and the vehicle groups.
The exact neuroprotective mechanism of the curcumin is unknown . In English literature to date, the curcumin was used in only one report as a neuroprotective agent after SCI. In this previous report, it has been shown that curcumin shows its effect by inhibiting apoptosis . In the present study, biochemical effects of curcumin after SCI were demonstrated for the first time in the literature. The pathological findings supported the biochemical analysis results. In addition, the functional measurements were compatible with the findings of the studies by Lin et al. To clarify the protective effects of curcumin on SCI, studies with larger sample size and different dosage of curcumin are needed. In addition, awareness of the ultrastructural changes in the spinal cord following curcumin treatment may shed light on its protective effects.
In conclusion, curcumin treatment after experimental SCI in rats improves early functional, biochemical, and pathological results almost as much as methylprednisolone does. According to our literature review, this is the first study demonstrating the neuroprotective effect of curcumin biochemically after SCI. Although the results of the present study have provided some interesting data confirming the useful effect of curcumin after SCI, further studies are under way to verify this mechanism in detail and to clarify the therapeutic window, dosage, and duration of treatment.