Salvianolic Acid B Ameliorates Motor Dysfuntion in Spinal Cord Injury Rats

Purpose: To evaluate the effect of salvianolic acid B (Sal B) treatment on the motor function of spinal cord injury (SCI) rat. Methods: SCI rats were modelled by contusion, and then received 10 mg/kg Sal B, or methylprednisolone, or phosphate-buffered saline (PBS) intraperitoneally daily for 4 weeks, two hours after the trauma occurred. During the treatment, footprint analysis (FA), inclined plane test (IPT), Basso-Beattie-Bresnahan (BBB) rating and Schnell Swim Test (SST) were used for estimating the recovery of motor function. At the same time, tissue edema was measured by wet-dry weighting, and the secretion of cytokines were indirectly quantitated by real time polymerase chain reaction (qPCR). Results: Primarily, Sal B group rats scored higher by FA, IPT and BBB rating. Further statistical analysis of comprehensive SST data from Student-t test indicates that Sal B can significantly ameliorate motor dysfunction after a 4-week treatment (p < 0.05) as well. Furthermore, Sal B decreased water content of the edema by 16.5 % during the first week, and sharply downregulated the transcription of interleukin 6 (IL-6) and tumor necrosis factor (TNF-α) 28- and 16-fold, respectively. Conclusion: The beneficial effect of motor function recovery was observed in SCI rats following intraperitoneal administration of Sal B.


INTRODUCTION
SCI refers to injuries occurred to the spinal cord, it may be caused by primary injury due to trauma, and secondary injury due to the cascade of cellular and molecular events that after trauma [1,2].Depending on where the spinal cord and nerve roots are damaged, the symptoms can vary widely, from pain to paralysis.In the United States, the incidence of SCI has been estimated to be about 40 cases per million population per year, and there are around 265,000 individuals living with SCIs at present with over 80% occurrence in males [3].The prevalence of SCI is approximately 60,000 per year in China according to a recent report [4].
A traditional Chinese medicine, Salvia miltiorrhiza has been widely used in Asian countries for the treatment of cardiovascular and cerebrovascular diseases [5].Currently, two major components extracted from the root of S. miltiorrhiza are Tanshinone IIA and salvianolic acid B (Sal B).The antioxidant Sal B has been shown to improve functional recovery in braininjured rats and provide neuroprotective effects in some experimental models of cerebral ischemia [6,7].Recently, Deng et al. reported that Sal B improved motor function are partially due to inhibition of increased TNF-α in the damaged spinal cord and exhibits neuroprotective effects [8,9].
The present study was designed to determine whether Sal B could be reliably used for motor function repairing in SCI rats.With the contusion animal models, a series of tests were used for evaluating the capacity of Sal B treatment on SCI rats.By multiple-factor comparison, we confirmed the effectiveness of the Sal B treatment and shown how much extent it can be improved critically in terms of the motor function.Except for direct damage to the spinal cord from trauma, the secondary injuries are important for SCI treatment.Therefore, we also measured tissue edema and changes of cytokines in the spinal cord contusion model.

EXPERIMENTAL Animals and drugs
Healthy Sprague Dawley rats (40), weighing 220 to 250 g (7 weeks old, male), were supplied by the Animal Center of Dalian Medical University, China.All the animals were housed at 25 °C in polycarbonate cages, 5 rats fed in one cage under controlled lighting (12 h light-dark cycle).Rats had free access to standard rodent water and chow throughout the experiment.Protocols were conducted in accordance with the latest Guide for the Care and Use of Laboratory Animals [10].The standard chemical solution of Sal B was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing).PBS solution and methylprednisolone were purchased from Selleck (Shanghai, China).

Establishment of SCI model and treatment
Sprague Dawley rats (30) were subjected to acute spinal segment injury by contusion [11].Briefly, the rats were intraperitoneally anesthetized using 0.4 % chloral hydrate (40 mg/kg).The injury was introduced by impact force (20 g, 2.5 cm) to the segments T11 and T12 of spine, damage in which leading to paralysis of the leg muscles above and below the knee in human.The success of SCI model establishment was affirmed with the posterior limbs of retraction-like flutter and flaccid paralysis.The other 10 rats without SCI were housed in two cages as healthy control.For each group, animals were treated as folllowing: 10 rats were intraperitoneally administered with 10 mg/kg Sal B, 10 rats with exactly the corresponding volume of PBS, and other 10 rats with 10 mg/kg methylprednisolone, respectively.
All treatments began at 2 hours after injury, and lasted for 4 weeks with 24 hours interval.

Test of tissue edema
All forms of injury can cause spinal cord edema, further decreasing blood flow and oxygenation.Edema is one of the secondary events which might aggravate a primary injury and there are reasons to believe that compounds, like SalB, might be useful to ameliorate the adverse effect.As one of the most important traditional herbal medicines, it is widely used in clinic in China, Japan, and other countries for the treatment of coronary artery disease and other cardiovascular diseases.The wet and dry weight method was used to evaluate the extent of tissue edema [11].

Evaluation of behavioral defect
A variety of methods have been used to assess animal functional recovery after SCI.FA is one of the widely used non-invasive and quantitative method [12].A series of at least six sequential steps were used to determine the mean values for measurement in a 5-point scoring system.The observation was conducted at the end of every week.IPT has been used routinely in models of SCI to clearly quantify the functional deficits due to major trauma to the central nervous system, which was modified from previously reported methods [13].This technique requires the rat to maintain its position for 5 seconds on a movable plane when the angle is adjusted by 1° inclination from 40° to a maximum of 55°.The maximum angle at which the rat can keep its position was recorded.The test was conducted once a week.BBB locomotor scale, the most widely used open field test has been accepted as a valid way to assess locomotor function after SCI in the rat [14].All the rats were allowed to adapt for 5 min after they were moved out to an open field.The movement of each animal was recorded by a digital camera and subsequently scored by the evaluators, who were blind to the treatment of the injuries.A more comprehensive method, SST was proposed to measure motor function and recovery in spinal cord injured rats [15].In this work, we measured: the velocity, forelimb strokes, tail movement, and the position of hindpaw.SST was conducted at the end of the treatment.

Cytokine detection by qPCR
Total RNA was extracted from injured tissue samples using the RNAeasy Kit (Qiagen, Germany) and 1 μg total RNA was used for cDNA synthesis with oligo dT and MMLV-RT purchased from Invitrogen.A total of 2 μL cDNA was subjected to mRNA quantification using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA).Briefly, quantitative PCR was performed using Roche LightCycler 480 for 40 cycles at 95°C for 10s, specific annealing temperature for 5 s and annealing at 72 °C for 10 s.Amplification specificity was checked using melting curve following the manufacturer's instructions.All experiments were performed twice in triplicate; all primers used were synthesized by Invitrogen (Shanghai, China) according to reference [16], sequence details are listed in Table 1.

Statistical analysis
The results are expressed as mean ± standard deviation (SD).Statistical comparison between control and the drug-treated groups were performed using Student-t test for in vivo data, P < 0.05 was set as the level of significance.

Ethical approval
This study was approved by the Animal Ethics Committee of Dalian Medical University, China.

Water content of edema tissue decreased after Sal B therapy
Three rats from the SCI group were randomly chosen for tissue edema test after 1, 3, and 7 days Water content in the spinal cord significantly decreased after 3 days in the model group treated with 10mg/kg Sal B (Table 2; P <0.05).Methylprednisolone can ameliorate tissue edema as well, though less effective than Sal B. After 7 days injection of Sal B, the edema tissue of SCI rats contained only 66.8 % water, which is about 10 % less than that for PBS-treated rats.

Sal B ameliorates motor deficits evaluated by FA, IPT, BBB, SST
Sal B-treated rats showed higher score after one week, and the scores increased over the following 3 weeks (Fig 1).This gait demonstrated a significant improvement in the Sal B-treated rats compared with the controls.Obviously, the improvement is much greater after 3 weeks injection.Healthy rats can stay for 5 seconds on an inclined plane at an angle of 52°.However, the SCI rats treated with PBS as negative control after 1 week could only maintain their position at a much lower angle of, 42° exactly, while, the injuried rats that received 10 mg/kg Sal B demonstrated much stronger capacity to move on the plane at an angle as high as 49° (Fig 2).Even, following treatment (weeks 2 -4) steadily enhanced this beneficial effect, but the improvement was clearly not as distinct as the first week.On the first day, all rats scored less than 4, indicating the contusion caused severe damage to motor function.And then, scores of all groups were increased greatly after treatment for 7 days (Fig 3).However, the trend becomes slower after two weeks for Sal B treated group even its score is still much higher than other groups.However, these two tests are too simple to illustrate the thoroughly measurable effectiveness on SCI treatment.Therefore, BBB locomotor rating which is almost exclusively used in this situation was used for evaluation.Indeed, the BBB scale is the most popular method for tracking functional outcome in moderate or mild injury models as well [19].In general, using this method produces consistent results.That is Sal B treatment improves motor function, and this beneficial effect is evidently stronger in the former two weeks.When considering more parameters, the effectiveness of Sal B is limited within the cure-period (4 weeks), while BBB rating cannot produce a correct assessment of forelimb-hind limb coordination.Some researchers successfully used the regularity index (RI) to determinate the functional recovery after SCI [20], the calculation of RI is much complicated as it relating to a few factors.Rats can swim naturally, so swimming can be used to obtain objective results in the analysis of motor function after various types of spinal cord lesions.For mild to moderate bilateral dorsal lesions, the assessment of velocity and forelimb strokes can be sufficient to obtain reliable results which correlate with the extent of the lesion or with the degree of recovery after treatment [21].Swimming also allowed the novel observation in rats of muscle spasms in spinal cord injured rats during the course of recovery [22].
After SCI, one common phenomenon is tissue edema.It seems Sal B eliminates the water content effectively after 3 days.The same result was also obtained in other study [9].A complex cascade of pathophysiological processes rapidly damage the nervous tissue after initial tramua.Inflammation is one of the major secondary pathologies following SCI [23], and severe inflammatory responses in the injured spinal cord destroy neurons, leading to exacerbate neurodegeneration [24].Inflammation contributes to secondary damage partly through over-release of cytokines, such as TNF-α, which enhance permeability of endothelial cells and facilitate trans-endothelial migration of activated leukocytes to the injured area [25,26].Virtually all nucleated cells, but especially endo/epithelial cells and resident macrophages are potent producers of IL-1, IL-6, and TNF-α [27].Some cytokines (such as IL-6) can increase up to 1000fold during trauma or infection.The expression of IL-6 is sharply increased in the acute stages after spinal cord injury and it may serve as a factor strongly inducing the differentiation of neural stem cells into astrocytes [28].In our experiment, the lower IL-6 concentration is related to less SCI as well.Sal B may suppressed the secondary injury by inhibiting the transcription of IL-6 and TNF-α, as proposed by other reaserch [9].
Methylprednisolone was the first neuroprotective drug to be discovered, and is the current standard in treatment although its efficacy has been called into question [29].We used this chemical as a positive control, to compare the effectiveness of Sal B. Other studies reported various drugs for the experimental cure of SCI [30,31]; however, we believe that Sal B is a good competitor considering its multiple usage and the results obtained in this paper.

CONCLUSION
Sal B is a promising promoter of rat motor dyfunction recovery after estimation by footprint annlysis, inclined plane test, BBB rating and SST analysis.At the same time, Sal B treatment can ameliorate tissue edema and downregulate the transcription of proinflammatory cytokine IL-6 and TNF-α.

Fig 1 :
Fig 1: Weekly FA of rats after treated with different chemicals.Values are scored as following standards: 0, no constant dorsal stepping or hindlimb dragging; 1, at least three toes in three footprints; 2, rotation of the feet more than double; 3, no signs of toe dragging but foot rotation; 4, no signs of rotation but more than one visible heel print; 5, no visible heel prints.PBS, Sal B and Methylprednisolone are SCI rats treated with them respectively.Error bars indicate SEM; n=10 animals per group.Key: ◇ = PBS group; ■ = Sal B group; ▲ = methylprednisolone group.

Fig 3 :
Fig 3: Temporal effect of different chemicals on motor disturbances after SCI by BBB rating.Key: ◇ = PBS group; ■ = Sal B group; ▲ = methylprednisolone group.

Fig 4 :Figure 5 :
Fig 4: SST of motor function recovery after treatment in contusion SCI rats.(A) Swimming velocity.The mean of three runs obtained from normal rats was taken as 100 % (30 frames).(B) Forelimb strokes.A maximum of 2 forelimb strokes per 60 cm swimming distance was considered the normal value, representing 100 %.One more forelimb stroke decreases the performance by 2 %. (C) Tail movement.A score of 4 represents normal tail movements in unlesioned rats, while a completely paretic tail with very rare twitching was given a score of 1, and a total absence of movement scores 0. (D) Hindpaw position.The normal swimming position was assigned 4, while the most lateral position was given a score of 1. Norm: normal rats without SCI.**p < 0.05; ***p < 0.01

Table 2 :
Injured spinal cord water content (%) Rats were allowed to remove the injured spinal cord segments.These segments were immediately weighed and dried in an oven maintained at 100 o C for at least 72 h until the dry weight became constant.* *Significant