Palm vitamin E reduces locomotor dysfunction and morphological changes induced by spinal cord injury and protects against oxidative damage

Spinal cord injury (SCI) occurs following different types of crushes. External and internal outcomes of SCI are including paralysis, cavity, and cyst formation. Effects of dietary derived antioxidants, such as palm vitamin E on central nervous system (CNS) encourage researchers to focus on the potential therapeutic benefits of antioxidant supplements. In the present study, experiments were carried out to evaluate the neuro-protective effect of the palm vitamin E on locomotor function and morphological damages induced SCI. Seventy-two male rats (Sprague-Dawley) were randomly divided into four groups: sham (laminectomy); control (supplemented with the palm vitamin E at a dose of 100 mg/kg/day); untreated-SCI (partial crush, 30–33% for 20 sec); treated-SCI (partial crush, 30–33% for 20 sec supplemented with the palm vitamin E at a dose of 100 mg/kg/day). The treatment with the palm vitamin E significantly improved the hind limb locomotor function, reduced the histopathological changes and the morphological damage in the spinal cord. Also, the palm vitamin E indicated a statistically significant decrease in the oxidative damage indicators, malondialdehyde (MDA) level and glutathione peroxidase (GPx) activity in the treated-SCI compared to the untreated-SCI.

Histopathology. The untreated-SCI and treated-SCI were sacrificed on day 3, 7, 14, 21 and 28 PO. The cavity formation could be observed from day 7 to 14 which followed by the cyst formation after the serial longitudinal sectioning. In the microscopic assessment, the cavity volume size was decreased between the day 3 (0.1788 ± 0.04 mm 3 ) Figure 1. Changes in the body weight in rats at pre-test and post-operation time. *p < 0.05 considered to be significantly different values compared to sham group in the respective day.
to day 14 (0.1142 ± 0.08 mm 3 ) in the treated-SCI group and then inclined between day 14-28 was slightly from 0.1142 ± 0.1 mm 3 to 0.1735 ± 0.04 mm 3 . While in the untreated-SCI group, the graph depicts a slight increase at day 3-14 (0.1454 ± o.1 to 0.2103 ± 0.08 mm 3 ) and then sharply increased to day 28 (0.3277 ± 0.06 mm 3 ) (Fig. 3A,B). The identification of the haemorrhage areas was possible in the lesion epicentre instantaneously after SCI (Fig. 4A) which resulted in increasing of the SC haemorrhage. Also, the cavity formation and oedema were observed within first week after the injury. As it is listed in Table 1, there were significance (p < 0.05) differences of the lesions size between the rostral-to-caudal and lateral-to-lateral in the treated-SCI and untreated-SCI groups at any point of the time. The lesion volume sizes were determined by lateral-to-lateral, rostral-to-caudal and specimen thickness (5 µm) measurement (Fig. 4A). Using Image J software, the yielded sums of the volume size in the treated-SCI group and untreated-SCI group were 2.731 mm 3 and 7.906 mm 3 , respectively. As shown in Fig. 4B, the untreated-SCI group cavity volume size was 2.89 times higher than that for the treated-SCI group. Due to different times of studies, the measurements of the size cavity were not the same. Figure 4C depicts the sums of volume cavity sizes of the treated-SCI and untreated-SCI groups.
In the sham group, there were no hind limb locomotor dysfunction immediately after anaesthesia till to the end of the study (day 28). Also, in the microscopic observation, the intact grey and white matters with distinct border lines were found (Fig. 5). In the present study, another side microscopic observation showed agglomeration of the erythrocyte in the cavity-shape area at day 3 in the treated-SCI group, which was much higher compared to those observed in the treated-SCI group. Till day 21, in the untreated-SCI group, abnormal neural cells with pale nucleus, more glial cells and also some cavities could be observed (Fig. 6).
Lipid peroxidation (MDA) analysis. A statistical significant (P < 0.05) difference of the tissue MDA levels which were calculated using the protocol inside the kit was observed in the untreated-SCI, treated-SCI and sham group at all points of time. The MDA level was found much higher in the untreated-SCI (4.9 µm) compared to that in the treated-SCI (1.2 ± 0.06 µm) and sham (2.1 ± 0.00 µm) groups (Fig. 7).
Tissue Glutathione Peroxidase (GPx) analysis. The tissue GPx activity was found markedly different in the treated-SCI, untreated-SCI and sham groups. The GPx activity increased in both treated-SCI (57 ± 1.5 nm/ ml) and untreated-SCI (111 ± 3.2 nm/ml) animals compared to that in the sham (38 ± 1.5 nm/ml) group. The spinal cord antioxidant marker, such as GPx concentration elevation, could be a sign to of oxidative stress caused by injury which will be discussed further in the paper (Fig. 8).

Discussions
In the present study, the oral supplementation of palm vitamin E at a dose of 100 mg/kg/day reduced the neurological deficits in the spinal cord injury. This dose is almost equivalent to the normal human dose (50-200 mg/ day). Published studies have proved that palm vitamin E at a dose of 100 mg/kg showed neuro-protective and also neuro-regenerative activity due to its antioxidant properties 19,[21][22][23][24] .
Our data showed the treated-SCI rats displayed less cavity formation, suggesting that palm vitamin E possessed anti-trauma and antioxidant properties which are in parallel to those of pervious findings 25,26 . The method used in current study for causing the crush injury, which led to the trauma, was the application of Inox no. 2 forceps. The reason of applying this method is because only a forceps is required which is cheap and easy to use. Therefore, it is introduced as a more effective candidate than other methods, such as weight dropping or balloon compression 27,28 . Behavioural improvement after palm vitamin E treatment. Locomotor functions were monitored by the BBB score among all the groups one day before (pre-test) and after operation. 28 days after treatment, the mean BBB score in the treated-SCI was significantly higher than that in the untreated-SCI group (mean ± SD, *p < 0.05) considered to be significantly different values versus sham group.
Although the body weight changes were stable throughout week 3 to 4 PO, but a slight drop in weight was observed during the first week after the surgery. Some factors such as stress, SC trauma, and loss of blood or fluid during the surgical procedure are being suggested which could be related to the weight loss 29 . Another word, the main important factors causing weight lost through the first week was highly suggested due to the rat's hind limb locomotor dysfunction or paralysis and also metabolic parameters affected by the operation. The pathophysiological implications of such dysfunctions were represented by the increase in production of the reactive species which are extremely aggressive to the surrounding tissues 30 .
In current research, the treated-SCI group showed gain weight compared to the untreated-SCI group which might be due to the antioxidant effect of the palm vitamin E. Bedreag et al., in 2014 30 showed that ill patients with spinal cord injury were more prone to oxidative stress due to the multitude of the biological dysfunctions. There are many biologically active substances with antioxidant properties of palm vitamin E that have proved to be able to reduce oxidative stress which contribute to boost up the recovery after SCI 29 . The preserved body weight in the sham group was associated with absence of any operation on the spinal cord and also having sufficient nutrition 31,32 . Since, the spinal cord crush limited access to enough food or water, therefore, the inconsistency in the weight loss was observed 32 .
We found that the BBB score varies on day 3, 7, 14, 21, and 28 PO in the untreated-SCI, treated-SCI, and sham groups. In the sham group, only the vertebrae of thoracic area were removed, while the SC was completely intact. Thus, the animals with no damage in the spinal cord did not show any changes in their BBB score during 28 days. It was reported that laboratory animals with incomplete contusion SCI reached maximal locomotor recovery within the first few weeks' post-injury 33,34 . So, the SC possessed the main role in performing locomotor tasks. There are some evidences to suggest that SC trauma, except the external signs and symptoms, was dependent to several plasticity caused contusion, hemi-contusion, hemi-section and etc. 33,34 . The results of our current study showed that there were no changes in the BBB score in the treated-SCI and untreated-SCI groups during the first 7 days. Afterward, the different BBB score in the named groups started to increase significantly, while on day 28 the BBB score for the treated-SCI group reached a score of 15 but the untreated-SCI group gained a score The volume of the cavities in treated-SCI group decreased from day 3 to day 14 and then slightly increased within the rest days. In untreated-SCI group the volume of the cavity was slightly increased, then significantly increased in day 28. Data expressed as mean ± SD. *p < 0.05. of 11. It is found that the palm vitamin E having potent antioxidant properties is able to exert protective effects by the reduction of oxidative damage. After SCI, a variety of oxygen radicals released from the cells which led to cell damages. The free radicals were scavenged by the palm vitamin E which contributed to reduction of the cell damages resulting in improvement of the locomotor function 35 . The main behavioural differences between the treated-SCI and untreated-SCI groups could be anticipated as the extensive movement of all three hind limbs joints (ankle, knee and hip) which were observed in all the studied animals. Although, various methods such as graded hemi-contusion, contusion, hemi-section etc 31,33 . have been applied to evaluate locomotor function in cervical and thoracic parts of spinal cord, but almost similar results in locomotor function have been reported 27,36 .
A large variety of semi-quantitative and whole scale based on the locomotor response of rats are currently being used to evaluate locomotor rating scale after SCI, such as BBB Locomotor Rating Scale 7,8 , 5-pt Tarlov Table 1. Measurement of the rostral-to-caudal and lateral-to-lateral of the cavities in the treated-SCI and untreated-SCI groups within 28 days after spinal cord injury. Data are expressed as mean ± SD. PO: post operation.  grid-walk test 38 , narrow beam test 39 and inclined plane test 40 . It is claimed that those SCI rats which display alternative locomotor functions following injury could not be classified by qualitative visual based analysis. Therefore, many quantitative procedures were extended to evaluate SCI motor function 41,42 . Guertin in 2005 43 recommended a new semi-quantitative assessment method called average combined scores (ACOS) which calculated basic locomotor-like activities based on the spinal reformation at the sub-lesion level. Compared to the BBB scale, the ACOS method was more multifaceted, time-and energy-consuming. ACOS uses video and electromyogram (EMG) recording to estimate locomotor recovery function which was more regarded as scientific quantitative assessment.
The various lesions of the rostral-to-caudal and lateral-to-lateral cavity sizes per time points are listed in Table 1. The lateral-to-lateral, rostral-to-caudal and specimen thickness measurements determined the cavity and/or lesion volume size per unit. There were significant changes in the cavity volumes and lesions size between the treated-SCI and untreated-SCI during the period of study, which could be due to the neurodegenerative effects and antioxidant activity of palm vitamin E.
Alpha-tocopherol and alpha-tocotrienol act as membranous antioxidants in mouse fibroblast cells 22 . In our study, the improvement of the BBB score in the treated-SCI group might be explained by the regeneration of the SC tissues affected by palm vitamin E which was also alluded in the literatures for organs such as, heart and cardio-vascular system, liver and coagulation system, brain and CNS, blood brain barrier and etc. 11 .
The study performed by Girard S, et al. 44 in utilizing multiple behavioural tests for evaluating functional recovery and residual function after SCI in the rodent models supports our findings 45 . It is obvious that different level of SC trauma is considered as an important factor for developing future therapeutic approaches.  On the microscopic assessments during all the intervals post-injury, the cavity lesions were smaller in the treated-SCI group compared to the untreated-SCI rats. Since palm vitamin E acts as a potent antioxidant agent in mitochondrial injury recovering, therefore, it might improve survivability upon glutamate toxicity due to its capability of oxidative stress elevation 10,46,47 . Researchers are using different types of vitamin E, such as tocotrienol-rich fraction (TRF) or alpha-tocopherol for treatment of different tissue damages. Xueling et al. 19 showed that TRF could have better curing effects on different tissue abnormalities compared to tocopherol types.
Antiserum inhibitor of nitric oxide synthesis 12 , Nitro steroid NCX 1015 48 intra-peritoneal (IP) citocholine 49 and Curcumin 50 because of their antioxidant effect, are able to reduce SCI following by trauma. However, many promising attempts have been made to reduce the signs of SCI, but none have yielded reliable effects.
It is suggested that free radicals and subsequent lipid peroxidation may delay the treatment progress of SCI-induced by any type of trauma. Antioxidants such as those from palm vitamin E family reduce trauma after SCI. Thus, motor dysfunction after SCI is shown to be affected by alpha-tocopherol 51,52 .
The MDA levels increased in the untreated-SCI, while the palm vitamin E supplementation delayed this process in the treated-SCI group. literature study showed that treatment with palm vitamin E attenuated the IL-1β in the injured SC, and also decreased the oxidative stress infiltration of neutrophils after SCI 52 . We evaluated the therapeutic effects of the palm vitamin E on the SC specimen using spectrophotometer and noticed that serious neuronal loss diffuse haemorrhagic areas were accumulated in SCI animals at 24 hours after operation. It was suggested by Miriam et al. 53 that IL-1β played a harmful role in the progress of post-traumatic damage. Such effects could be reduced by blocking its signalling pathways. We followed some of the probable mechanisms by which the palm vitamin E attenuated neurological damage. It was shown that SCI enhanced penetration of oxidative stress, which was determined by an increase in lipid peroxides and diminution of enzymatic (GPx) antioxidants in the tissue. The palm vitamin E exerted dominant antioxidant effect in conjugation with a decrease of the free radical-derived products (MDA), and improved the recovery in SC or CNS after injury 54 . In the untreated-SCI group, an increase in the free radical activity was observed which in turn could enhance the levels of the MDA and GPx. In the treated-SCI group, a significant decrease was observed in the antioxidant activity compared to the untreated-SCI and sham groups which was in response to the oxidative stress followed by injury. This findings are supported by the work reported in the literature that palm vitamin E dietary supplementation showed brain and neuronal cells protective effect against dysfunction brain injury and Alzheimer disease 19,55 .

Conclusion
It is concluded in vivo studies that on 28 days of the treatment of SCI with the palm vitamin E could have the maximum reduction in spinal cord cavity and cyst formation. Four weeks after the treatment, an increase regenerating in spinal cord is observed leading to functional recovery of the essentially paralyzed hind limbs. The spinal cord injury lead to paralysis, mostly to the hind limb. It is found that the palm vitamin E is a promising impact on the injured spinal cord. These beneficial effects, together with the biological safety and other properties such as antioxidant effect suggests that this approach may have clinical applications in the treatment of SCI.

Methods and Materials
Animals. Eight-week-old male Sprague-Dawley rats weighing 250 ± 50 g were used (n = 72). The animals were housed in an environment with a 12 hours' dark/light phase at the standard condition of temperature (21 ± 1 °C) and controlled humidity (50 ± 10%). The standard laboratory food and water were given ad libitum and each wire cage housed two rats. The experimental processes in this study were approved by the Animal Care Committee of the Faculty of Medicine's Animal House, Institutional Animal Care and Use Committee; University Malaya (UM), according to the guideline and policy of University Malaya; regarding the care and use of animals for scientific purposes with the reference number.
The number of animals was calculated using Power analysis based on the pervious study from our lab 56 .
Surgical procedures. The rats were anesthetized using a mixture of 100 mg/kg of kertamil (100 mg/ml ketamine, Australia) and 10 mg/kg of xylazil (20 mg/ml xylazine, Australia) through intra-muscular (IM) injection. 70% alcohol was used to clean the skin and then, the back thoracic area were shaved for further experiments. The spinal column was exposed to T11-L1 and, for the transverse apophyses, the paravertebral muscles were removed. For the sham group, the SC was opened but no injury was inflicted, and for the treated-SCI and untreated-SCI groups, to manage the partial crush injury, Inox no.2 modified stopper forceps were used. The partial crush was administered at the T12 level of the spinal cord. The saline solution was applied to clean the wound and then for repair the fascia, muscles, and skin, the absorbable sutures and clips were used, respectively. During surgery, the body temperature was maintained at 37 ± 0.5 °C constantly, while a hot plate was applied to preserve the same temperature during the post-operative (PO) period 57 .
Post-injury Care. After the operation, a 1 ml of the lactated ringers' solution was injected subcutaneously (SC), and the topical antibiotic was applied to the skin at the incision site. A 5% Baytril (Bayer, Korea) IP injection was given at a dose of 2 mg/kg/day for one week after the surgery. After the operation, an analgesic was administered as well as it needed (Buprenorphine 5 µg/kg every eight hours). The food was always accessible in the food pellets were placed on the bottom of each cage. The gastrointestinal and hydration functions were monitored every day. During one week after surgery, the rats were weighed every day; after which they were weighed every week. Post-operative care comprised manual bladder expression twice a day until the bladder function returned. Palm vitamin E and diets supplementation. The four groups were fed with a standard rat chow. The animals were given access to food and water ad libitum. The control and treated-SCI groups were supplemented with 100 mg/kg/day palm vitamin E (through an oesophageal feeding tube) for 28 day according to the protocol published by Yao et al. 58 .
Behavioural analysis. Open field task. At pre-test time (one day before surgery), the rats underwent locomotor function evaluation in the experimental chamber. During pre-test evaluation, all animals environmental condition such as temperature, humidity and light, should be stable similar to experiment days. Each animal's locomotor activity in both pre-test and experiment time was evaluated by two examiners in an open field system over four minutes. The open field was a plexi-glass circle chamber with 100 × 100 cm dimensions and the walls measuring about 9-11 cm, placed at a room temperature with normal lighting and a quiet environment.
Hind limb locomotor function. The rats were also assessed 1 and 3 days after surgery, after which their assessment was carried out once a week until day 28 after the operation. The certain functional behavioural conditions were measured using the BBB locomotor rating scale. This scoring system is explained by Basso et al. 8 . The specific components of functional behaviour were assessed such as tail position, trunk and abdomen position, coordination, toe clearance, stepping, limb mobility, and paw position. In the BBB scale, a score of 21 is an indication of normal hind limb locomotor function (complete mobility) and a score of 0 is a sign of lack of natural hind limb movement (complete immobility).
Histopathological assessment. Tissue processing. At the time of sacrifice, a mixture of xylazine (0.3 mg/ kg, xylazine, Australia) and ketamine (0.7 mg/kg ketamine, Australia) were used to deeply anaesthetize the rats via IM. Each rat was put in their prone position and the thorax was shaved. Using scissors to cut both sides of the sternum, the cardiac area was opened and the left ventricle (cardiac apex) was perfused using a butterfly angiocath perfusion catheter. The vena cava or left atrium were cut and the area perfused with about 50 ml normal saline (until/clear). This was followed by about 150 ml of 4% formalin in millioning's phosphate buffer, applied at the rate of 350-400 ml/min for the peristaltic pump. The vertebrae were then removed and placed in 4% formalin in millioning's phosphate buffer overnight at an approximate temperature of 4 °C, constantly and then in the following day, the spinal cord was removed from the vertebrae.
Haematoxylin and Eosin staining. To prepare the serial sections (5 µm), a section (1 cm) from rostral-to-caudal of the lesion epicentre was placed in paraffin in tissue-Tek; tissue embedding machine with laboratory cryostat. Haematoxylin and Eosin staining (H and E) and light microscopy were used to carry out the histopathological evaluation of the SCI for each sample. Then the cavity area for the lesion of the sham, treated-SCI and untreated-SCI group, traced and then calculated. Quantitative image analysis procedure. The photographs of the spinal cord specimens were placed under the microscope (Nikon, Japan) and then analysed. The injury border was drawn, and the lesion area of rostral-to-caudal, lateral-to-lateral cavity and cyst formation measurement were calculated for each sample using the NIS-Elements AR 3.2 software connected to the microscope eclipse (Nikon eclipse 80i, Japan). In the treated-SCI and untreated-SCI groups, the area of the lesion was discerned by the presence of necrosis, inflammatory cells, cavity and also cyst formations. The lesions sizes were measured according to two parameters: rostral-to-caudal and lateral-to-lateral in cavity and cyst formation.
Lipid peroxidation (MDA) analysis. MDA is a natural product of lipid peroxidation. Lipid peroxidation is a well-known mechanism in animal and plants cell damage. It is also used to suggest oxidative stress in cells and tissues 61 . The SC specimen from the sham group, only in area of SC which removed vertebrae and in treated-SCI and untreated-SCI groups at the injured area (1 cm rostral and 1 cm caudal from lesion area) were removed 24 hours after scarify of rat at day 28 and immediately stored at −80 °C until use. The specimens were homogenized separately in the cold assay buffer (on ice) and then centrifuged at 12,000 rpm for 30 min at 4 °C. MDA level was assessed with the thiobarbituric acid reactive substances TBARS (TCA Method) assay kit; Cayman chemical, USA, item No. 700870) according to the manufacturer's instructions.
Glutathione Peroxidase Assay (GPx). Glutathione peroxidase (GPx) catalyses reduction of hydro-peroxides including hydrogen peroxide, by decreasing glutathione function to protect cell from damage. The spinal cord specimen from the sham, treated-SCI and untreated-SCI groups, 24 hours after scarify of rat at day 28, were removed and homogenized separately in 5 volumes of the cold assay buffer (50 mM Tris-HCL, pH 7.6, containing 5 mM EDTA)/g) 62 . The homogenised specimen was then centrifuged for 15 min at 10000 g at 4 °C and the supernatant was removed for GPx according to the manufacturer's protocol (Cayman chemical, USA, item No. 703102).
Statistical analysis. The data were analysed using SPSS 11 for Windows. All outcomes are expressed as means ± SD. One-way analysis of variance (ANOVA) with Tukey's post hoc analysis was used to compare differences among the groups. If the P value was less than 0.05, it was considered statistically significant. The data collected from the experiments was recorded and analysed using SPSS software (version 19.0, Inc., Chicago, IL, USA).