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The efficacy of alpha-tocopherol in functional recovery of spinal cord injured rats: an experimental study

Abstract

Objective:

The objective of this study is to examine the effects of the alpha-tocopherol on rats with spinal cord injury (SCI).

Setting:

Research Center, Sultan Bin Abdulaziz Humanitarian City, Riyadh, Kingdom of Saudi Arabia.

Method:

Female Sprague–Dawley rats weighing 180–220 g were anesthetized with chloral hydrate (450 mg kg−1 body weight) by intraperitoneal injection and laminectomy was performed at the T 7–8 level leaving the dura intact. A compression plate (2.2 × 5.0 mm) was loaded with a weight of 35 g placed on the exposed cord for 5 min to create SCI. The subjects were divided into three groups of eight rats each. Group 1 served as control (SCI+saline); whereas groups 2 and 3 served as test groups, alpha-tocopherol was given orally in doses of 1000 mg kg−1 body weight for group 2 and 2000 mg kg−1 body weight for group 3, respectively. Daily activities were recorded in the activity cage for 14 days post-operatively.

Results:

At day 1 (baseline, 24 h after the surgery), there was no significant difference between mean motor scores of all groups. After day 1, the three groups showed continuous improvement in motor score; such improvement was maintained throughout the duration of the study with different levels for each group. By the end of the study (day 14), groups 2 and 3 showed statistically significant improvement in the mean motor score compared with group 1 (P<0.05). However, no significant difference was observed between test groups 2 and 3 by the end of the study.

Conclusion:

The results suggest that the administration of alpha-tocopherol may have reparative effects for SCI because of its antioxidant effect.

Introduction

Spinal cord injury (SCI) is a devastating and common neurological disorder that has profound influences on modern society from physical, psychosocial and socioeconomic perspectives.1 It is a leading cause of permanent disability in young adults, resulting in partial or complete loss of motor and sensory functions below the lesion site.2 Currently, treatment options are limited, but significant advances have been made in understanding the pathophysiology of SCI.2 This decade has been labeled as the decade of the Spine to emphasize the importance of SCI and other spinal disorders.1 SCI caused by trauma mainly occurs in two mechanisms, such as primary and secondary injury. Secondary injury after the primary impact includes various pathophysiological and biochemical events.3 Furthermore, SCI results in loss of sensory and motor functions because injured axons do not regenerate and neurons that die are not replaced.4 Chronic SCI can lead to an insidious decline in motor and sensory functions in individuals even years after the initial injury and is accompanied by slow and progressive cytoarchitectural destruction.5 The extent of morphological remodeling after SCI is, however, not understood.4 However, the adult central nervous system is capable of considerable anatomical reorganization and functional recovery after injury. Functional outcomes vary greatly, depending on the size and location of injury, type and timing of intervention, and type of recovery and plasticity evaluated.6 Spontaneous regeneration of injured axons or plastic rearrangements of spared fiber systems after SCI in mammals is very limited.7 The main reasons for this poor spontaneous repair capacity seem to be the insufficient growth response of neurons to injury, the growth-inhibitory components of the adult central nervous system tissue, and the formation of cysts and scar tissue at the injury site.7 Attempts to overcome local barriers by grafting peripheral nerve bridges,8 Schwann cells9 or olfactory ensheathing cells10 have led to regenerative fiber growth and in some instances to behavioral recovery in animal models of SCI, although the mechanistic understanding of this recovery remains incomplete because of the complexity of these interventions.10 Varying degrees of neurological function spontaneously recover in humans and animals during the days and months after SCI.11 Studies have suggested that the generation of free radicals and subsequent lipid peroxidation have been proposed to contribute to delayed tissue damage after traumatic SCI.12 Ubiquinols (reduced coenzyme Q), ascorbate (vitamin C) and alpha-tocopherol are endogenous antioxidants; decreases in tissue levels of these compounds may therefore reflect ongoing oxidative reactions.12 Many studies have been conducted to understand the effect of alpha-tocopherol on compression injury in the spinal cord of rats. The motor disturbance induced by SCI was greatly reduced by alpha-tocopherol supplementation.13 After injury, the spinal cord evoked potentials that showed greater recovery of both amplitude and latency in the alpha-tocopherol-supplemented group than in the control group.14 The aim of this study was to examine the effects of alpha-tocopherol on spinal cord injured rats.

Materials and methods

Animals

Adult female Sprague–Dawley rats weighing 180±220 g were used in the study. The animals were housed in polycarbonate cages with sawdust bedding. They were kept in a temperature-controlled room (23±2 °C) and maintained on 12-h light/dark cycles, with free access to standard laboratory food (Grain Silos and Flour Mills, Riyadh, Kingdom of Saudi Arabia) and tap water. The protocol of animal studies was approved by the Research and Ethics Committee of Sultan Bin Abdulaziz Humanitarian City (SBAHC), Riyadh, Saudi Arabia.

Drugs

Alpha-tocopherol was used as an antioxidant (Sigma-Aldrich, Steinheim, Germany) and chloral hydrate was used as an anesthetic agent (Merck, Darmstadt, Germany).

Dosing and testing

Female Sprague–Dawley rats weighing 180–220 g were anesthetized using chloral hydrate (450 mg kg−1 body weight) by intraperitoneal injection for the rats and laminectomy was performed at the T 7–8 level leaving the dura intact. A compression plate (2.2 × 5.0 mm) loaded with a weight of 35 g was placed on the exposed cord for 5 min. Animals were divided into three groups of eight rats each. The animals in group 1 served as control (SCI+saline), whereas the rats in groups 2 and 3 were subjected to SCI as mentioned above and received alpha-tocopherol orally in the doses of 1000 mg per kg body weight for group 2, and 2000 mg per kg body weight for group 3, respectively, for 14 days.

Behavioral studies

Animals were carefully observed for any behavioral abnormality before the daily administration of drugs. The infrared beam-array activity cage-7431 (Ugo Basile Biological Research Apparatus, Comerio, Varese, Italy) was used to monitor the activity of the animals. The activity cage basically relies on horizontal and vertical sensors. The movements of the animal inside the cage interrupt one IR beam per second. The beam interruptions are counted and recorded by the electronic unit, which enable the examiners to assess and analyze the animal activity.

Statistical analysis

The activity scores were analyzed by one-way analysis of variance (ANOVA). Tukey–Kramer multiple comparisons test was used for comparing the activity score with the test groups. P-values <0.05 were taken as statistically significant.

Results

Body weight (g)

Group 1 showed a gradual decrease at day 2 (177±2.21 g, P<0.01), day 3 (174±1.98 g, P<0.01), day 4 (169±2.21 g, P<0.001), day 5 (170±2.46 g, P<0.001), day 6 (168±2.19 g, P<0.001) and day 7 (170±2.25 g, P<0.05) as compared with the baseline value (184±2.69 g). However, there was a gradual increase in the body weight from day 8 (171±2.32 g, P>0.05) until the end of the study (day 14) (187±2.7 g, P>0.05) as compared with the baseline value (184±2.69 g) (Table 1).

Table 1 The effect of SCI and different treatments on animal's body weight (g)

Group 2 showed a gradual decrease starting at day 2 (177±1.64, P<0.01) until day 8 (166±1.24 g, P<0.001) as compared with the baseline value (184±1.43 g). However, there was a steady and continuous increase in the body weight at day 9 (170±1.38, P<0.001) that was maintained until the end of the study (day 14) (194±1.39 g, P<0.05) as compared with the baseline value (184±1.43 g). Compared with group 1 (control group), group 2 showed a significant improvement at day 13 (P<0.01) and day 14 (P<0.05) (Table 1).

Group 3 showed a gradual decrease of body weight at day 2 (181±2.15 g, P<0.01) until day 6 (169±2.31 g, P<0.001) as compared with the baseline value (188±2.13 g) (Table 1); however, and as shown in group 3, there was a gradual increase in the body weight at day 7 (172±3.58 g, P<0.001) that was maintained through the following days until the end of the study (day 14) (198±0.97 g, P<0.05) as compared with the baseline value (188±2.13 g). Compared with group 1, group 3 showed a significant improvement at day 10 (P<0.05), and this significant improvement was maintained until the end of the study (day 14) (P<0.01) (Table 1).

Vertical movement score (mean±s.e.m.)

Group 1 showed a gradual improvement in vertical movement at day 4 (286±29, P>0.05), and such improvement was maintained at all time points until the end of the trial period (day 14) (856±83, P<0.001) as compared with the baseline value (219±21) (Figure 1).

Figure 1
figure1

The effect of alpha-tocopherol (1000 mg kg−1) in SCI rats. Values are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001. *P-values vs baseline-Tukey–Kramer multiple comparisons test. Groups compared: 1 vs 2, #P<0.05, ##P<0.01.

Group 2 showed the same trend in the positive outcome, and there was an improvement in vertical movement at day 3 (250±11, P>0.05). Such movement was clearly shown and maintained at all time points throughout the study period until the end of the study (day 14) (1126±83, P<0.001) as compared with the baseline value (226±10). However, by comparing groups 1 and 2 together, group 2 showed marked improvement at day 8, and this movement did increase gradually and showed statistically significant difference by the end of the study (days 13 and 14) (Figure 1).

Group 3 showed a gradual improvement in vertical movement at day 2 (267±28, P>0.05), and this movement was continuously maintained through day 3 until the end of the study (day 14) (1105±78, P<0.001) as compared with the baseline value (238±23). However, compared with the control group, group 3 showed marked improvement at day 2, which was maintained throughout the study period and was observed to be statistically significant observed by the end of the study (day 14) (P<0.05) (Figure 2).

Figure 2
figure2

The effect of alpha-tocopherol (2000 mg kg−1) in SCI rats. Values are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001. *P-values vs baseline-Tukey–Kramer multiple comparisons test. Groups compared: 1 vs 3, P<0.05.

Horizontal movement score (mean±s.e.m.)

Group 1 showed a gradual improvement in horizontal movement at day 3 (230±18, P>0.05), this movement was steadily continuous throughout the trial period until the end of the study (day 14) (646±38, P<0.001) as compared with the baseline value (223±20) (Figure 3).

Figure 3
figure3

The effect of alpha-tocopherol (1000 mg kg−1) in SCI rats. Values are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001. *P-values vs baseline-Tukey–Kramer multiple comparisons test. Groups compared: 1 vs 2, #P<0.05, ##P<0.01.

On the other hand, group 2 showed a gradual improvement in horizontal movement score at day 3 (198±10, P>0.05) until the end of the study (day 14) (890±62, P<0.001) as compared with the baseline value (238±12). Compared with the control group, group 2 showed marked improvement at day 7 and was maintained until the end of the study with statistically significant changes observed at day 13 (P<0.01) and day 14 (P<0.05) (Figure 3).

Group 3 did maintain the same improvement trend as in group 2 in horizontal movement starting at day 3 (275±37, P>0.05). This improvement was also continuous through the study period until the end of the study (day 14) (915±55, P<0.001) as compared with the baseline value (238±21). However, by comparing with group 1, it is clearly obvious that the improvement shown in group 3 was markedly better than the results shown in the control group. This improvement was noticed at day 2 and was maintained at all time points of the trial, which was statistically significant by the end of the study (day 14) (P<0.001) (Figure 4).

Figure 4
figure4

The effect of alpha-tocopherol (2000 mg kg−1) in SCI rats. Values are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001. *P-values vs baseline-Tukey–Kramer multiple comparisons test. Groups compared: 1 vs 3, †††P<0.001.

Discussion

Traumatic injury to the spinal cord typically results in axonal damage and cell death and leaves individuals with varying degrees of functional impairments. The extent of these impairments is dependent on both the severity of the injury as well as the level at which the injury occurred.2 It is well recognized that the evolution of secondary tissue damage spreads away from the injury epicenter, incorporating tissue both rostral and caudal to the primary lesion with increasing functional deficits. The fact that damage continues to develop over time in the days and weeks after acute SCI provides an opportunity to intervene. Neuroprotective strategies aimed at preventing damage arising from secondary injury processes provide some hope for tissue sparing and improved functional outcome. This, in conjunction with the fact that current treatment options are limited, hastens the need to find novel therapeutic agents.1

In this experimental study, SCI reduced the body weight of the rats for the first 7 days followed by a slow recovery (Table 1). This is in accordance with previous studies that reported that SCI reduced animal body weight. This is partly due to the great stress exerted on the body at the time of the initial trauma, so the body's metabolism works faster to provide energy and nutrients to help in healing the body and fighting infections. As a result, it helps in decreasing the weight of the animals.15, 16, 17 The results of this study showed that there was gradual recovery over a period of 14 days after SCI compared with baseline readings. These observations are in agreement with previous findings.13, 18

Previous studies were conducted to evaluate the efficacy of alpha-tocopherol on compression injury in the spinal cord of rats. The motor disturbance induced by SCI was greatly reduced by alpha-tocopherol supplementation.13, 18 After injury, the spinal cord evoked potentials that showed greater recovery of both amplitude and latency in the alpha-tocopherol-supplemented group than in the control group.13, 18 Acute SCI produces tissue damage that continues to evolve days and weeks after the initial insult, with corresponding functional impairments. Reducing the extent of progressive tissue loss (neuroprotection) after SCI should result in better recovery.19 It has been suggested that the generation of free radicals and subsequent lipid peroxidation has been proposed to contribute to delayed tissue damage after traumatic SCI. Ubiquinols (reduced coenzyme Q), ascorbate (vitamin C) and alpha-tocopherol are endogenous antioxidants; hence decreases in the tissue levels of these compounds may therefore reflect ongoing oxidative reactions.12 In this study, the motor disturbance induced by SCI was greatly reduced by alpha-tocopherol. This observation is in agreement with other investigators, who also reported that alpha-tocopherol may have reparative effects on SCI.13, 18 Animals that received 1000 mg kg−1 of alpha-tocopherol showed significant improvement in the vertical and horizontal movements when compared with the control animals. Furthermore, when animals were treated with 2000 mg kg−1 of alpha-tocopherol for 14 days, it produced an improvement from day 2 and was clearly shown by the end of the study (days 13 and 14). However, there is no significant difference between groups 2 and 3. In conclusion, the results of this study clearly indicate that alpha-tocopherol increases the motor activity of SCI rats, which suggests that alpha-tocopherol may have reparative effects for SCI because of its antioxidant effect.

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Acknowledgements

We would like to express our great thanks and sincere appreciation to Dr Majed Al-Kasabi, the Director General of the Sultan Bin Abdulaziz Al-Saud Foundation for his valuable support to the Research Center.

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Correspondence to S Al-Mubarak.

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Al Jadid, M., Robert, A. & Al-Mubarak, S. The efficacy of alpha-tocopherol in functional recovery of spinal cord injured rats: an experimental study. Spinal Cord 47, 662–667 (2009). https://doi.org/10.1038/sc.2009.22

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Keywords

  • spinal cord injury
  • functional recovery
  • neurological disorder

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