Liang LIa,b, Feng LIa,c, Jian YINb, Bo PANb, Qing-Ping YUb, Xiong CAIa, Hui-Yong HUANGa*, An CHENb
a.Provincial Key Laboratory of TCM Diagnostics,
b.Key Discipline of Anatomy and Histoembryology, Hunan University of Chinese Medicine, Changsha, China
c.School of Dentistry, University of California Los Angeles, California, USA
Hui-Yong HUANG, M.D., Professor, Provincial Key Laboratory of TCM Diagnostics, Hunan University of Chinese Medicine, Science and Education Park, Yuelu District, Changsha 410208, China. Research direction: digitalization and intelligentification of Traditional Chinese Medicine.
Objective: To establish a rat model of Spinal Cord Injury (SCI) with pathopattern of Qi-deficiency and Blood-stasis (QDBS) in Traditional Chinese Medicine (TCM), and then assess its feasibility. Methods: Swimming exhaustion method and Rubrospinal Tract (RST) transection operation had been combined to establish the rat model of SCI with pathopattern of QDBS in TCM. Macroscopic representation, including the body weight, food intake and tongue scores, was recorded. Behavioristics was scored with spontaneous vertical exploration test. Hemorrheology was analyzed by the hemorheological analyzer and morphous of spinal cord were observed. After the reverse demonstration by Buyang Huanwu Decoction (BYHWD), feasibility of the model had been assessed. Results: The circumstance in the QDBS group was in line with the QDBS syndrome, showing body weight, food intake, and utilization rate of forelimbs decreased, and tongue scores increased. However, the data above in the BYHWD group were superior to the QDBS group after 35 d (P<0.05). Compared with the control group, the whole blood viscosity and hematocrit increased in the QDBS group (P<0.05), but the deformability index of RBC decreased (P<0.05). All data above in the BYHWD group were close to the control group. An eminence or induration appeared in the injured spinal cord, which was suspected to be vacuole or scar. HE staining showed that the ratio of R/T in the BYHWD group was higher than that in the QDBS group (P<0.05). Conclusion: Swimming exhaustion method combined with RST transection operation can successfully establish a rat model of SCI with pathopattern of QDBS in TCM.
- 2.Materials and methods
- Funding information
- Competing interests
Spinal cord injury (SCI) is expected to cause a variety of social problems because of its high incidence and disability rate, and the high cost of treatment and low mortality rate associated with it [1,2]. Although there are no curative methods for SCI available at present, a large number of experimental and clinical studies have shown that Traditional Chinese Medicine (TCM) has a unique effect on SCI . In TCM theory, SCI is attributed to the Qi deficiency and blood stasis (QDBS) syndrome , which states that central nervous system injury can lead to different forms of dysfunction, such as hemiplegia, facial distortion, aphasia, and angular salivation. The mechanism underlying these effects may be related to damage to the DU meridian, weakness of Qi, stagnation of blood, impaired passage across meridians and collaterals, and malnutrition of the muscles and vessels. In this condition, both “Qi deficiency” and “blood stasis” exist simultaneously. The pathogenesis of SCI in TCM theory is very complex; therefore, establishment of a feasible animal model with the QDBS pathopattern is a prerequisite for SCI research in TCM.
2.Materials and methods
The Buyang Huanwu Decoction (BYHWD) contained the following ingredients: 120 g Huang Qi (Radix Astragali), 6 g Dang Gui (Radix Angelica Sinensis), 4.5 g Chi Shao (Radix Paeoniae Rubra), 3 g Di Long (Lumbricus), 3 g Chuan Xiong (Chuanxiong Rhizoma), 3 g Hong Hua (Flos Carthami) and 3 g Tao Ren (Semen Persicae). All herbal components used in BYHWD were obtained from the LBX Pharmacy Co., Ltd (Hunan province, China), and identified by an expert of the Department of Pharmacy, Hunan University of Chinese Medicine. They were then extracted according to the standard methods outlined in the Pharmacopoeia of the People’s Republic of China . The mixture was decocted in boiling water for 45 min, concentrated, and vacuum-dried to form a paste containing components equivalent to 2 g of crude extracts per gram. This BYHWD paste was diluted with distilled water to a concentration of 1.0 g/ml, which was stored at 4°C until further use.
2.2Animals and experimental design
This was a randomized, controlled animal study performed at the Anatomical Research Laboratory of Hunan University of Chinese Medicine (Changsha, Hunan province, China). A total of 24 male Sprague-Dawley rats (weight, 200-220 g; age, 9-10 weeks) were provided by the Laboratory Animal Center of Hunan University of Chinese Medicine (License no. SYXK [Xiang] 2013-0005; Changsha, Hunan province, China). The rats were separately housed in plastic cages at 22-25°C and 50%-70% humidity under a constant light-dark cycle and were allowed free access to food and water.
The animals were randomly divided into the control, QDBS, and BYHWD groups (n = 8 per group). Models with the QDBS pathopattern were established in the QDBS and BYHWD groups. To achieve this, the rats were subjected to the forced swimming test over 21 d, followed by rubrospinal tract (RST) transection. The duration of the experiment was 49 d. Macroscopic assessments, behavioral analysis, hemorheologic evaluation, and morphological assessment of the spinal cord were performed (Figure 1). The BYHWD group received intragastric perfusion of 25.65 g of the crude drug/kg body weight  from day 22, while the control and QDBS groups received an equal amount of normal saline. On day 49, all experimental animals were sacrificed. All procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China .
2.3Establishment of the Qi-deficiency state
The Qi-deficiency state [8,9] was established using the forced swimming test, which was performed by placing the rats in a glass cylinder (diameter, 100 cm; height, 60 cm) containing water (20 ± 1°C) with a depth of 40 cm with no escape platform. After 3 days’ adaptive swimming training, all rats underwent the forced swimming test (once a day, 1 h each time) over a 21-d period. The test involved forcing the rats to swim continuously till exhaustion, which was identified on the basis of the following observations: action incoordination, immersion up to the tip of the animal’s nose, and inability to remain afloat for at least 10 s. When the rats were subsequently rescued, they gasped sharply with slightly closed eyes; their bodies were swaying and they raised their hind limbs slowly, with some rats being unable to move for a long time because of spastic hind legs. These findings confirmed the state of swimming exhaustion. The animals were allowed to rest for 1-2 minutes and then made to swim again, for a total duration of 1 h each day.
2.4Establishment of the blood-stasis state
The blood-stasis state was established by performing RST transection after the 21-d forced swimming tests. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (4 ml/kg body weight). After separating the superficial structures under an operating microscope, the spinal process of C2 could be easily identified by the multiple muscles attached to it radically (Figure 2A, 2B). The erector spinae on the right side of this spinal process was removed to clearly show the vertebral arches of C3 and C4, and the ligamenta flava between C3 and C4 were transected to expose the spinal cord (Figure 2C). After identification of the dorsal root entry zone and the midline of the spinal cord, a small incision was made through the dura mater, and the right dorsolateral funiculus of the spinal cord was transected with a sharp blade. This lesion completely transected the lateral funiculus (including the RST), and partially injured the ipsilateral ventral funiculus and gray matter but left the dorsal columns intact. The dura mater was then closed with interrupted 10-0 silk sutures, and the muscles and skin were closed in layers . After the surgery, the rats were maintained on heating pads, observed closely until waking, and then returned to their home cages. The operations were considered successful if the animal’s right forelimb could not move normally, flexing and adhering closely to the trunk, and the forepaw could not open (Figure 2D).
The body weight and food intake of all rats were recorded on days 0, 7, 14, 21, 35, and 49. The tongue scores were obtained after intraperitoneal injection of 10% chloral hydrate (4 ml/kg body weight) on days 0, 21, 35, and 49. The tongue color and sublingual collaterals were observed, and the scores were evaluated as described previously .
To assess locomotor function recovery, the forelimb utilization rate during spontaneous vertical exploration was examined as described previously [7,11,12], on days 0, 24 (3 d after SCI), 35, and 49. Rats were placed individually in a transparent glass cylinder (diameter, 18 cm; height, 24 cm), which encouraged the use of forelimbs for spontaneous vertical exploration, for a 5-min period. The rats would spontaneously stand up in the narrow space and touch the inner wall of the cylinder frequently with their forelimbs for exploration. The following behaviors were scored within the 5-min period: frequency of use of both forelimbs (N) and independent use of the right (injured) forelimb (n) for touching the inner wall of the cylinder. The data were presented as the utilization rate of both forelimbs ([N/(N+n)]) and the right (injured) forelimb ([n/(N+n)]). The assessments were made by two researchers who were blinded to the interventions.
Blood samples were obtained from all rats before sacrifice, and the following parameters were measured with an automatic hemorheologic analyzer according to the manufacturer’s instructions: the high (200/s)/mid (30/s)/low (1/s) shear rates for whole blood viscosity, hematocrit, deformability index of red blood cells (RBCs), and electrophoresis time of RBCs.
2.8Morphological observations of the spinal cord
After collection of blood samples, the rats were anesthetized again with a dose of 10% chloral hydrate (4 ml/kg body weight), followed by perfusion of 0.9% saline solution (100 ml) through the heart and perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4 at 4°C; 150 ml). A long segment of the spinal cord, 4 cm in length, was dissected from the vertebral column, such that the lesion area was at the midpoint of the segment. The dissected segment was post-fixed for 2 h at 4°C and subsequently immersed till sinking in a graded series of sucrose solutions (10%, 20%, and 30%).
Gross appearance: The external features of the injured area in the spinal cord were observed.
Microscopic appearance: The C2 segment was selected, embedded in OCT compound, sliced at a thickness of 20 μm with a constant freezing microtome at -20°C, and subjected to hematoxylin-eosin (HE) staining. Four slices from each rat were chosen to observe the overall shape of the spinal cord under a microscope (40×). The ratio of the right half (injured) to the total area (R/T) of the spinal cord was calculated using Image Pro software.
Statistical analysis was performed using SPSS software, version 19.0 (SPSS, Inc., Chicago, IL, USA). Measurement data were expressed as mean ± standard error (SEM) values. Two-way analysis of variance (ANOVA) was used to analyze the body weight, food intake, tongue score, and behavioral data. One-way ANOVA was used to analyze differences in hemorheologic data and the R/T ratios. P values less than 0.05 were considered to indicate significance.
Body weight and food intake: The intergroup differences in body weight and food intake values began to increase from day 14 (P < 0.05); the values were remarkably different over the period of model establishment, with the differences being statistically significant (P < 0.05). In the BYHWD group, the body weight and food intake increased after intragastric administration of BYHWD over the last 4 weeks (Figure 3).
Tongue scores: The tongue scores in the QDBS and BYHWD groups were higher than those in the control group on days 21, 35, and 49 (P < 0.05); however, no statistically significant difference was observed between the scores in the BYHWD and QDBS groups on day 21 (P > 0.05). In comparison with the QDBS group, the BYHWD group showed lower scores on days 35 and 49, i.e., after BYHWD administration (P < 0.05) (Figure 4).
3.2Spontaneous vertical exploration
The utilization rates of both forelimbs and the right forelimb were 40%-60% before RST transection. On day 24, the rates in the QDBS and BYHWD groups were not significantly different from those in the control group (P < 0.05). On day 35, the rates in the QDBS and BYHWD groups were lower than those in the control group (P < 0.05), with the BYHWD group showing higher values than the QDBS group (P < 0.05). On day 49, the utilization rates increased significantly in the BYHWD group, but the right forelimb utilization rate was still lower than that in the control group (P < 0.05); the utilization rates in the QDBS group did not show a remarkable recovery, and the values were lower than those in the other two groups (P < 0.05) (Figure 5).
The whole blood viscosity, hematocrit, and RBC deformability index in the QDBS group were significantly different from the corresponding values in the control group (P < 0.05); however, only the low shear rate value for whole blood viscosity was significantly different between the BYHWD and control groups (P < 0.05). Compared with the QDBS group, the BYHWD group showed significantly different values for the high and low shear rates for whole blood viscosity, RBC deformability index, and RBC electrophoresis time (P < 0.05) (Table 1).
3.4Morphology of the spinal cord
Gross appearance: In the control group, both the dura mater and spinal cord were intact, and each segment of the spinal cord had the same color and luster. In contrast, in the QDBS and BYHWD groups, the dura mater was incomplete in the lesion area; the pia mater became adherent to the spinal cord and could not be separated easily; and an eminence or induration was present on the local surface of the spinal cord (Figure 6A, 6B). If the spinal cord was dissected out directly without perfusion with saline solution and paraformaldehyde, the siltation of blood stasis in the local area was observed obviously (Figure 6C).
Microscopic appearance: HE staining showed that the right half of the spinal cord was intact in the control group, the white matter was dense and clear, and all the tracts were arranged neatly. However, in the QDBS group, the whole spinal cord had atrophied, and the area of the right half had decreased. Large quantities of scattered myelin sheaths and cavities could be seen in the completely impaired right posterolateral funiculus; the R/T ratio was lower than that in the control group (P < 0.01). The findings in the BYHWD group were similar to those in the QDBS group; while the R/T ratio in the BYHWD group was higher than that in the QDBS group (P < 0.05), it was still lower than that in the control group (P < 0.05) (Figures 7, 8).
The establishment of a QDBS pathopattern has been regarded as a major challenge among scholars. Most researchers initially focus on Qi deficiency, establishing a corresponding model with a single factor or compound factors. Some of them have used the forced swimming, forced running, or sleep deprivation methods [13,14,15], based on the TCM theory that over-exertion results in Qi exhaustion; some preferred to use the starvation method [16,17], which is based on the theory that Qi originates from food; and others applied a combination of hunger, fatigue, panic, cold, medicines, and other factors [18,19]. Although there are multiple methods, they fundamentally simulate only the cause of the disease, without simulating and locating the disease itself. Therefore, these methods cannot facilitate assessments in line with the progression of the disease, resulting in wasted experimental effort.
In this study, a model of SCI with the QDBS pathopattern was established by integrating disease identification and syndrome differentiation. The Qi-deficiency state was first established with the swimming exhaustion method, based on the theory that over-exertion results in Qi exhaustion. The long-term exhaustion induced in the forced swimming test resulted in Qi deficiency. TCM theory states that Qi is the power of blood flow and that it pushes the blood to move freely along meridians and in vessels; thus, long-term Qi deficiency will eventually lead to blood stasis and then the generalized state of QDBS . We then performed RST transection to ensure the state of blood stasis in the local area . The spinal cord is located in the vertebral canal, which is equivalent to the DU meridian in TCM. Local damage to the spinal cord is similar to injury to the DU meridian, which leads to blockage of channels and impairs the passage of Qi and blood. Sluggish or no movement of Qi and blood results in the state of blood stasis.
The reasons for choosing RST transection to establish the state of blood stasis are as follows: (1) the RST originates from the red nucleus in the midbrain, and nearly all the fibers (99%) descend downwards to the contralateral posterolateral funiculus of the spinal cord . The unambiguous position, concentrated fibers, and clear boundary allow for easy selective transection on one side of the RST, leaving most of the remaining spinal cord intact. (2) RST is believed to regulate the fine locomotor skills of the forelimbs in rats, thereby facilitating behavioral observations . (3) The traditional SCI model is the “striking model,” which is usually directed at the lower thoracic segments ; in this process, most of the lamina of the vertebral arch needs to be removed violently, causing harmful trauma, excessive bleeding, and loss of reflective micturition. The animals that undergo this process require rigorous postoperative care, failing which they may die due to severe uroschesis, urinary tract infection, or pressure ulcers . In contrast, RST transection does not injure the lamina of the vertebral arch and causes less trauma and bleeding ; only the posterolateral funiculus of the spinal cord is transected. This approach is superior to the traditional model because it preserves the function of the urinary bladder, allows normal food intake and defecation, requires normal levels of postoperative care, and has a high survival rate. Only two rats died during our experiment, and the mortality rate was only 8.3%. Thus, the swimming exhaustion method was combined with RST transection to create a reasonable SCI model with the QDBS pathopattern.
This model was evaluated according to the theory of “Biological Representation” used in TCM syndrome models and the idea of “Treatment according to the syndrome diagnosis” . In this context, “Biological Representation” refers to the macroscopic characteristics of the QDBS pathopattern in animals, including the activity level and fur, eyeball, nail, ear, nose, lip, and tongue color. The concept of “Syndrome diagnosis” represents all kinds of symptoms induced by the QDBS pathopattern, while the term “Treatment” refers to the administration of the Chinese herb decoction. In other words, the feasibility of the animal model can be confirmed on the basis of the reversion caused by treatment with the corresponding Chinese herb.
In addition to these aspects, the blood stasis should be verified to evaluate the rationality of the model. Microcirculatory abnormalities have been proven to be one of the basic pathological changes in blood stasis syndrome . The diagnostic criteria for blood stasis syndrome established by Chinese scholars in 1986 have been widely used in clinical practice, and these criteria state that hemorheologic parameters should not be ignored . There are numerous hemorheologic parameters, such as blood viscosity, RBC deformability index, RBC aggregation, hematocrit values, blood coagulation factor, and plasma fibrin levels. In general, hemorheologic assessments can clarify the concentration, viscosity, coagulation, and aggregation of blood, and changes in blood viscosity and flowability are important factors in the diagnosis of blood stasis syndrome .
In this study, macroscopic findings, behavioral data, hemorheologic findings, and morphological data of the spinal cord were obtained. The feasibility of the model was assessed on the basis of the reversion caused by BYHWD administration. The following aspects were primarily evaluated: first, assessments of body weight, food intake, and tongue scores were performed to evaluate the macroscopic characteristics of QDBS syndrome. Second, the data from spontaneous vertical exploration tests were used to determine locomotor function recovery of the injured forelimb, which indirectly revealed the effect of the interventions. Third, the hemorheologic assessments indicated the generalized state of blood stasis, while the morphology of the spinal cord illustrated the definite state of blood stasis in the local area. Finally, the reversion of the model was demonstrated using BYHWD, a famous prescription used for supplementing Qi and activating blood, to verify the rationality of the model.
After undergoing the forced swimming test for 21 d, the rats in the QDBS and BYHWD groups showed the characteristics of Qi deficiency to varying degrees, including shortness of breath, fatigue, laziness, poor appetite, weight loss, and a dim tongue. The body weight and food intake of the rats in the QDBS and BYHWD groups decreased in comparison with the values in the control group from day 14 (P < 0.05), but they increased gradually in the BYHWD group after administration of BYHWD. In comparison with the control group, the QDBS and BYHWD groups showed crimson or cyanotic tongue with petechiae and higher tongue scores (P < 0.05). However, in comparison with the QDBS group, the BYHWD group showed lower scores in the last 3 w (P < 0.05). These findings show that BYHWD can remarkably improve the generalized state of QDBS.
With respect to locomotor function recovery, the right forelimb could not move after the RST transection in both the QDBS and BYHWD groups, while 14 d later, it recovered gradually. The utilization rates of both forelimbs and the right forelimb in the BYHWD group were higher than those in the QDBS group, but still lower than those in the control group. These findings indicate that BYHWD can significantly recover forelimb locomotor function.
Hemorheological measurements showed that in comparison with the control group, the QDBS group had higher whole blood viscosity and hematocrit values and a lower RBC deformability index, which confirmed the establishment of the blood-stasis state. All hemorheological values in the BYHWD group were close to those in the control group after BYHWD administration, in accordance with the findings of Wang’s study . As for the morphology of the spinal cord, an eminence or induration, which could be vacuoles or a scar, appeared in the injured area, illustrating the obvious local state of QDBS. HE staining showed that the right half of the spinal cord had atrophied remarkably and the R/T ratio had decreased in the QDBS group, with the value being lower than that in the BYHWD group (P < 0.05). These data indicate that BYHWD can remarkably improve the local state of QDBS.
In conclusion, the swimming exhaustion method combined with RST transection can successfully establish a rat model of SCI with the QDBS pathopattern. The model can not only describe the neurophysiological changes and locomotor behavior in SCI but can also represent the QDBS pathopattern, which is in accordance with the theory of syndrome manifestation in TCM. This model overcomes the disadvantages caused by the short duration of effects and the strong self-healing ability in traditional methods, and the reversion of its effects can be demonstrated by administration of the corresponding Chinese herb decoction. Thus, it is a feasible and stable model.
This work was supported by grants from the National Natural Science Foundation of China (No. 81302899, 81373551 and 81603512) and the Key Science and Research Program of Hunan Department of Science and Technology (No. 2012TF-1005).
We are grateful for the support provided by the Institute of Diagnostics of Traditional Chinese Medicine and Department of Anatomy, Hunan University of Chinese Medicine, Changsha, China.
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结果 气虚血瘀组大鼠体重、摄食量和前肢使用率降低，舌象评分上升，符合气虚血瘀证的表现；35 d后BYHWD组各指标均优于气虚血瘀组。与正常组相比，气虚血瘀组全血粘度、红细胞压积上升（P<0.05），红细胞变形指数下降（P<0.05）；BYHWD组经过中药干预后各指标基本接近正常组。脊髓受损局部出现隆起、硬结，疑似空泡或瘢痕，HE染色显示大鼠脊髓冠状切片伤侧面积比率BYHWD组高于气虚血瘀组（P<0.05）。