Si-Di LI a, Yi-Hua SHENG a, Xin-Min LIU c, Zu-Guang YE b , *
bThe Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College
cInstitute of Chinese Materia Medica, China Academy of Chinese Medical Sciences
Pharmacologist, researcher, professor. Deputy Director and National Pilot Base of National Engineering Research Center for New Drug Development of Traditional Chinese Medicine and concurrently serves as Chairman of Professional Committee of Novel Drug Delivery System of Chinese Medicine Association of the World Federation of Chinese Medicine and Chairman of Professional Committee of Drug Administration and Intellectual Property Protection of Chinese Medicine. Editor in chief of Chinese Journal of Information on Traditional Chinese Medicine.
Objective: To investigate the ability of Tong Luo Jiu Nao (TLJN) Oral Solution to improve cognitive and gait deficits in a rat model of focal cerebral ischemia.
Methods: An intraluminal suture method was used to create a rat model of focal cerebral ischemia. After 4 weeks of drug intervention, water maze and gait analyses were conducted. Brain tissue was stained with hematoxylin and eosin and used to monitor and calculate the volume ratio of cerebral infarction in each group of permanent middle cerebral artery occlusion (pMCAO) rats.
Results: (1) Gait analyses: TLJN Oral Solution significantly improved the overall gait of the focal cerebral ischemic rat model. The improvement in walking speed might have been achieved by the shortening of the animals’ stride time. The shortening of stance time by TLJN in the cerebral ischemic animals might be due to the shortening of brake and propulsion times, while the shortening of stride time by TLJN in the cerebral ischemic animals might have been due to the shortening of stance time and swing time. (2) Water maze navigation test: high-dose TLJN Oral Solution significantly reduced the animals’ total swimming distance and duration and increased the proportion of swimming distance and time spent in the target quadrant, suggesting that TLJN significantly improved the cognitive deficit in the animal model. (3) High-dose TLJN significantly reduced the cerebral infarction volume ratio of the pMCAO rats.
Conclusion: This intraluminal suture method can reliably replicate a rat model of focal cerebral ischemia. TLJN Oral Solution improves the gait and cognitive function of the focal cerebral ischemia rat model to various extents.
- 1. Experimental Materials
- 2. Experimental methods
- 3. Statistical analyses
- 4. Experimental results
- 4.1 Water maze test
- 4.2 Gait analyses
- 4.2.1 Walking speed and average stride time
- 4.2.2 Stance time
- 4.2.3 Swing time
- 4.2.4 Brake time
- 4.2.5 Propulsion time
- 4.2.6 Duty cycle
- 4.2.7 Right hindlimb–left hindlimb–right forelimb–triple-limb support time
- 4.2.8 Right forelimb–left forelimb–right hindlimb–triple-limb support time
- 4.2.9 Swing speed
- 4.2.10 Homologous coupling between the left and right hindlimbs
- 4.3 HE staining
- 5. Discussion
- Funding Support
- Competing Interests
1. Experimental Materials
1.1. Experimental animals
Male 8-week-old Sprague-Dawley (SD) rats weighing 280–320 g were used in this study.
1.2. Drugs and reagents
TLJN Oral Solution was prepared by the Beijing University of Chinese Medicine, while chloral hydrate was obtained from Sinopharm Chemical Reagent Beijing Co., Ltd.
The equipment used in this study includes the following: Sartorious-110S analytical balance (0.0001-g increments; Sartorious, Germany ); steam sterilizer (Shinva Medical Instrument Co., Ltd .); a high-speed refrigerated centrifuge (HITACHI Company); and a real-time image detection and analysis processing system for the water maze and gait experiments (joint development by The Institute of Medicinal Plant Development of the Chinese Academy of Medical Sciences and Peking Union Medical College, China Astronaut Research and Training Center, and Beijing Xinhai Instrument and Technology Company).
2. Experimental methods
2.1. Animal groupings
Seventy male SD rats weighing 280–320 g were randomized into four groups: (1) sham surgery, 10; (2) cerebral ischemia model, 20; (3) cerebral ischemia model + low-dose TLJN Oral Solution (150 mg/kg), 18; (4) cerebral ischemia model + high-dose TLJN Oral Solution (300 mg/kg), 20, of which two were eliminated due to behavioral gait training failure.
2.2. Animal model creation
The intraluminal suture method was used to create a model of focal cerebral ischemia in the model animals. The intraluminal suture occlusion method reported by Longa et al. was used to establish a rat model of permanent focal cerebral ischemia as follows. The rats were fasted the night before surgery, but were allowed to drink water. The rats were anesthetized with 10% chloral hydrate (33 mg/kg) injected intraperitoneally and placed in the supine position. The tissue was separated in layers through a midline incision in the neck to expose the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The proximal end of the CCA and the ECA stump were permanently ligated. The distal end of the CCA was temporarily closed by a microvascular clip. A diagonal incision was made at the distal end of the CCA ligation, through which a nylon suture with a smooth, rounded tip was inserted. The suture advanced to the CCA bifurcation and to the skull through the ICA into the anterior cerebral artery to occlude the blood flow to the middle cerebral artery (MCA), leading to infarction in the territory around the MCA. The insertion depth of the nylon suture was 20 mm. The distal end of the CCA was ligated to fix the nylon suture and prevent bleeding. The incision was closed in layers. The same procedures were performed in the sham surgery group except for the nylon suture insertion depth being 10 mm. All incisions were sterilized with penicillin solution prior to closure. The rats were fasted for 12 h pre- and post-surgery. The rats were kept warm after the surgery.
2.3. Drug administration
The drug was administered intraperitoneally after surgery once the animals were completely awake. The corresponding volume of pure water was given to the sham surgery group and the model group. In the treatment groups, high-dose or low-dose TLJN Oral Solution was given for 4 consecutive weeks. The animals were euthanized after treatment and the tissues were obtained for study.
2.4 Experimental method
2.4.1 Morris water maze test
A Morris water maze test was conducted 4 weeks after surgery and drug treatment. The water maze for the rats consisted of a round pool 150 cm in diameter and 60 cm deep. Three animal entry points were marked on the wall of the pool, which was divided into four quadrants. One of the quadrants housed a round metal platform 10 cm in diameter and 30 cm deep in the center. The water level was 1 cm above the platform. The water maze temperature was maintained at 25°C ± 1°C. The fixed reference points outside the pool, such as a door, a window, and decorations, remained in the same position for every test. The water maze tests were divided into two phases. The first phase was a 5-day navigation test. Each day of the test, the rats were placed into the pool facing the wall at one of the four different marked entry points. The time taken to locate the platform (latency to locate platform) was recorded for 2 min. If the platform could not be located within that amount of time, the rat would be placed on the platform for 15 s. The latency to locate the platform was recorded for 2 min. The second phase was a spatial exploratory test. The platform was removed after the navigation test. The rats were placed into the pool facing the wall at one of three different entry points. The swimming distance within the target quadrant was recorded for 2 min.
2.4.2 Gait experiment
Seven days before the experiment, each animal was trained on the walkway three times per day to ensure they could run continuously from one end of the walkway to the other. The experiment began after the training period. The lighting system of the equipment was adjusted prior to the test to ensure appropriate lighting conditions to clearly capture the animals’ footprints in the image-capturing window. Each animal ran across the walkway three consecutive times, while the corresponding three gait videos were recorded by a high-frequency camera. Computer analysis software was then used to analyze the images and calculate the gait parameters. The gait experiment was performed at 2 and 4 weeks after surgery and drug administration to analyze > 60 parameters, such as stride time, stance time, step length, step width, average turning angle of the body, and average intensity.
2.4.3 Hematoxylin and eosin staining of brain tissue
Saline and 4% paraformaldehyde were injected through the left ventricular aorta. The tissue was fixed by 10% neutral formalin and marked with a cross. Routine paraffin embedding was performed on the brain tissue slides with a thickness of 2 mm. Each 5-μm section of brain tissue was mounted onto a glass slide. Hematoxylin and eosin (HE) staining was performed after paraffin dewaxing.
3. Statistical analyses
SPSS version 16.0 software was used to analyze the experimental results. All data are presented as mean ± SEM. Repeated measures analysis of variance was performed of the behavioral data with observation time as the within-subject variable and sham surgery and model groups as between-subject variables. Mauchley’s test was used to determine if the variables met the assumption of sphericity. If the variable violated the assumption of sphericity, Greenhouse–Geisser was used to adjust the degrees of freedom. If there was a significant difference between repeated measurements, a post hoc analysis was used and the least significant difference test was applied to compare groups. Furthermore, Pearson’s correlation coefficient was used to analyze the relationship between infarct volume and behavioral outcome. Values of p < 0.05 were considered statistically significant.
4. Experimental results
4.1 Water maze test
As shown in Figure 1, the total swimming distance of the model group was significantly longer than that of the sham surgery group on days 1–5 of the navigation experiment. The total swimming distance of the model group was not significantly different from that of the low-dose TLJN group, whereas the high-dose TLJN group displayed shorter total swimming distance than the model group on days 2 and 5.
As shown in Figure 2, the total swimming time of the model group was significantly longer than that of the sham surgery group on days 1–5 of the navigation experiment. Compared with the model group, the total swimming time of the low-dose TLJN group was shorter on day 4, whereas the high-dose TLJN group displayed significantly shorter total swimming time on days 1, 2, 4, and 5.
As shown in Figure 3, the percentage of total swimming distance in the target quadrant was significantly shorter in the model group than in the sham surgery on days 1–5 of the navigation experiment. Compared with the model group, the percentages of total swimming distance in the target quadrant of the high-dose TLJN group was longer on days 1, 4, and 5 of the navigation experiment.
As shown in Figure 4, the percentage of time spent in the target quadrant was significantly shorter in the model group than in the sham surgery group on days 1–5 of the navigation experiment. Compared with the model group, the high-dose TLJN group spent higher percentages of time in the target quadrant on days 2–5 of the navigation experiment.
As shown in Figures 5–9, the model group showed significantly increased total swimming distance, significantly shorter swimming distance and time spent in the target quadrant, and significantly reduced percentages of swimming distance and time spent in the target quadrant than the sham surgery group during the spatial exploration experiments. The total swimming distance was shorter in the high-dose TLJN group than in the model group.
4.2 Gait analyses
4.2.1 Walking speed and average stride time
At weeks 2 and 4 after surgery, the average walking speed of pMCAO rats was significantly reduced compared to that of the sham group (p < 0.01). The high-dose TLJN group displayed significant recovery in post-surgery weeks 2 and 4 (p < 0.05 and p < 0.01, respectively). At weeks 2 and 4 after surgery, the average stride time of the pMCAO rats was significantly longer than that of the sham surgery group (p < 0.01). Additionally, the high-dose TLJN group displayed a significantly shorter average stride time 4 weeks after surgery (p < 0.01).
4.2.2 Stance time
Two weeks after surgery, the stance time of the four limbs was significantly longer in the pMCAO rats than in the sham surgery rats (p < 0.01). Low- or high-dose TLJN did not significantly improve the stance time. At week 4, there was still a significant increase in stance time of the four limbs of the pMCAO rats (p < 0.01), whereas high-dose TLJN effectively reduced the stance time of the four limbs of the pMCAO rats (left forelimb, p < 0.05; left hindlimb, right forelimb, right hindlimb, p < 0.01).
4.2.3 Swing time
Two weeks after surgery, compared with the sham surgery group, the swing time of the four limbs was significantly longer in the pMCAO rats (left forelimb, right forelimb, p < 0.05; left hindlimb, right hindlimb, p < 0.01). The high-dose TLJN rats had significantly reduced swing time (p < 0.05). Four weeks after model creation, there was still a significant increase in the swing time of the four limbs of the pMCAO rats (right forelimb, p < 0.05; left forelimb, left hindlimb, right hindlimb, p < 0.01), whereas high-dose TLJN effectively reduced the swing time of the two left limbs and the right forelimb of the pMCAO rats (p < 0.05).
4.2.4 Brake time
Two weeks after surgery, the brake time of the left forelimb was significantly increased in the rats of the model group compared to those of the sham surgery group (p < 0.05) with no significant differences in the rest of the limbs. At post-surgery week 4, the brake time of all four limbs in the model group was significantly increased (left forelimb, left hindlimb, right forelimb, p < 0.01; right hindlimb, p < 0.05). After 4 weeks of drug administration, the low-dose TLJN rats showed significantly reduced brake time of the right forelimb (p < 0.05). In the high-dose TLJN rats, with the exception of the right hindlimb, the other three limbs showed significantly reduced brake time (left forelimb, right forelimb, p < 0.01; left hindlimb, p < 0.05).
4.2.5 Propulsion time
Two weeks after surgery, the propulsion time of the four limbs was significantly increased in rats of the model group compared to those of the sham surgery group (p < 0.01). There was no significant improvement in propulsion time of the four limbs in the high- and low-dose TLJN rats. Four weeks after surgery, the propulsion time of all four limbs remained significantly longer in the model group than in the sham surgery group (left forelimb, right forelimb, right hindlimb, p < 0.01; left hindlimb, p < 0.05). After 4 weeks of treatment, the high-dose TLJN rats showed significantly reduced propulsion time in all four limbs (left forelimb, right forelimb, right hindlimb, p < 0.01; left hindlimb, p < 0.05).
4.2.6 Duty cycle
Two weeks after surgery, the duty cycles of the two forelimbs were significantly increased (left forelimb, p < 0.05; right forelimb, p < 0.01). High- and low-dose TLJN did not significantly improve the duty cycles of the two forelimbs. Four weeks after surgery, the duty cycle of the two forelimbs remained significantly increased in the model group compared to the sham surgery group (p < 0.01). In addition, at treatment week 4, low-dose TLJN reduced the duty cycle of the right forelimb (p < 0.05), whereas high-dose TLJN reduced the duty cycles of both forelimbs (p < 0.05).
4.2.7 Right hindlimb–left hindlimb–right forelimb–triple-limb support time
At post-surgery weeks 2 and 4, compared with the sham surgery group, the right hindlimb–left hindlimb–right forelimb–triple-limb support time of the pMCAO rats was significantly increased (p < 0.01). In addition, at treatment week 4, the high-dose TLJN rats showed significant decreases in right hindlimb–left hindlimb–right forelimb–triple-limb support time (p < 0.05).
4.2.8 Right forelimb–left forelimb–right hindlimb–triple-limb support time
At post-surgery week 4, compared with the sham surgery group, the right forelimb–left forelimb–right hindlimb–triple-limb support time of the pMCAO rats was significantly increased (p < 0.01). In addition, at treatment week 4, the right forelimb–left forelimb–right hindlimb–triple-limb support time was effectively reduced in the high- and low-dose TLJN rats (p < 0.05).
4.2.9 Swing speed
Two weeks after surgery, compared with the sham surgery group, the swing speed of all four limbs of the pMCAO rats was significantly decreased (p < 0.01), and high- and low-dose TLJN did not significantly improve the swing speed of the four limbs of the pMCAO rats. Four weeks after surgery, the swing speed of all four limbs of the pMCAO rats remained significantly decreased (p < 0.01). In addition, high-dose TLJN effectively improved the swing speed of the left forelimb, right forelimb, and right hindlimbs of pMCAO rats (p < 0.05).
4.2.10 Homologous coupling between the left and right hindlimbs
At weeks 2 and 4 after model creation, compared with the sham surgery group, the model group showed significantly increased homologous coupling between the left hindlimb and the right hindlimb (p < 0.05). At treatment week 4, compared with the model group, the high-dose TLJN group displayed significantly reduced homologous coupling between the left hindlimb and the right hindlimb (p < 0.01).
4.3 HE staining
Ischemic stroke is a common disease with high mortality and morbidity rates that seriously threatens human health and affects patient quality of life. The study of ischemic stroke is an important topic in the medical field. The main effective components of TLJN are Zhi Zi Gan (Geniposide) and San Qi Zong Zao Gan (Panax Notoginsenosides). Zhi Zi Gan (Geniposide) is the main active ingredient of Zhi Zi (Gardenia), which, with reference to the terms used in traditional Chinese medicine, can clear the “heat and fire” that build up in the body, cool the blood, and have detoxifying effects. Studies have shown that Zhi Zi Gan (Geniposide) possesses anti-inflammatory and anti-coagulation activities and has detoxifying, antioxidant, and anticancer properties1,2. San Qi Zong Zao Gan (Panax Notoginsenosides) is the effective active ingredient extracted from the valuable Chinese medicine San Qi (Panax Notoginseng), the main effects of which are increasing blood circulation and stopping bleeding. The traditional Chinese pharmacopoeia Bencao Xinbian considers San Qi (Panax Notoginseng) invigorating. Modern pharmacology has confirmed that San Qi Zong Zao Gan (Panax Notoginsenosides) has many anti-inflammatory functions, such as eliminating free radicals, reducing lipid peroxidation, suppressing Ca2+ influx, inhibiting platelet aggregation, dilating blood vessels, and improving microcirculation3.
Studies to date on the mechanism of TLJN efficacy and the related animal research have focused on its pharmacodynamic mechanisms such as its effects on oxidative stress of the brain tissue in acute cerebral ischemia, protection of endothelial cells, and anti-inflammation. There are few reports on the long-term overall behavioral effects of TLJN in cerebral ischemic animals. There is also a lack of detailed reports on improvements in gait functions in animal models with cerebral ischemia. One of the reasons may be that, in many preclinical studies, the observation window for assessing the volunteers’ functions after cerebral ischemia was relatively narrow. The evaluation of injury within a short period after stroke has limited our understanding of the disease. This is often due to the lack of information on the long-term effects of treatment; additionally, our bodies may possess a transient reversible neuroprotection system, which leads to so-called false-positives. Furthermore, the impairments related to sensorimotor dysfunction after stroke often extend to several months or several years4,5. Our group has conducted relatively in-depth studies on the mid- to long-term gaits of cerebral ischemic animals. Therefore, we sought to investigate the ability of TLJN to improve the cognitive and gait functions of cerebral ischemic animals.
The gait analyses of this study showed that walking speed significantly increased and stride time significantly decreased when TLJN was administered to cerebral ischemic rats. This suggests that TLJN can significantly improve the overall walking functions of cerebral ischemic rats and that the improvement in walking speed may be achieved by a decrease in the animals’ stride time, the sum of stance time and swing time. This study also showed that TLJN significantly decreased the stance and swing times of the animals’ four limbs, suggesting that the decrease in stride time induced by TLJN in the cerebral ischemic animals may be due to the respective decrease in stance and swing times. Next, stance time can also be categorized into brake time and propulsion time. This study showed that TLJN significantly reduced the propulsion and brake times of the cerebral ischemic animals, suggesting that the decrease in stance time induced by TLJN in the cerebral ischemic animals may be due to the respective decrease in brake and propulsion times. Propulsion time and propulsion index are often used to describe the process of an animal’s limbs lifting off the ground and the gradual decrease in footprint surface area in contact with the ground during walking. These two indicators can reflect the ability of the animals’ limbs to kick start and propel the body forward; therefore, to a certain extent, they also reflect the way the animals use their force and the muscle strength of their limbs when they are walking. In comparison, patients with hemiparesis have a reduced ability to imitate swing and propulsion using their lower limbs6.
Muscle strength is a key factor affecting walking. The knee extensor strength of the limb that is on the same side of the injured limb is one of the most important factors of walking speed. In patients with hemiparesis, the strength of the triceps surae muscle is reduced, the knee joint is unstable, and the ankle mobility is poor. They also have lower-extremity muscle spasms and decreased balance, resulting in a reduced ability to the push the body away from the ground and propel it forward, eventually leading to abnormal walking7. Studies have also shown that walking speed was significantly correlated with lower-limb muscle strength in stroke patients7,8. This has been verified by the results from our study, which show that, in cerebral ischemic animals, the prolongation of time in which the limbs are in contact with the ground increases the propulsion index and eventually leads to changes in the force mode used by the limbs, which may be an important factor leading to reduced walking speed. The biological explanation of these results may be the significant reduction in the physical strength of the animals’ limbs. Therefore, the significant reduction in propulsion time in the cerebral ischemic animals caused by TLJN may have been achieved by improvements in the physical strength of the cerebral ischemic animals’ limbs.
In patients with hemiparesis, compared with the normal population, the absolute time with ipsilateral and contralateral single-limb support is increased, with a larger increase in that of the contralateral limb. However, the percentages of the absolute time with ipsilateral and contralateral single-limb support with reference to the overall stride time in patients with hemiparesis is less than that in normal people. The decrease in percentage is more obvious in the ipsilateral limb, as its weight-bearing capacity decreased and is compensated by the contralateral limb. The present study found that 2 weeks and 4 weeks after the induction of focal cerebral ischemia in rats, the duty cycles of the forelimbs were increased, whereas no significant increase in the duty cycles of the hindlimbs were observed. The reason may be that the cerebral ischemic animals try to increase gait stability by avoiding the use of the more seriously injured ipsilateral hindlimbs and provide compensatory support using the remaining limbs, mainly the forelimbs. In other words, the cerebral ischemic animals used the forelimbs to compensate for the hindlimbs, leading to a longer time of support by the forelimbs compared with the hindlimbs. TLJN can significantly reduce the compensatory stance time of the forelimbs, suggesting that it may improve the gait function of the injured limb, thereby significantly reducing the stance time of the contralateral limbs.
Studies have shown that in patients with hemiparesis, compared with the normal population, the absolute double-limb support time is increased and the percentage of time spent with double-limb support also increases. The prolongation of double-limb support time may increase gait stability. In this study, the double-limb support time was decreased in the cerebral ischemic animals, whereas triple- and quadruple-limb support times were increased. These observations also suggest that the animals increase the multi-limb support time to compensate for the decreased gait stability. TLJN can significantly reduce the increased triple-limb support time, suggesting that the cerebral ischemic animals rely significantly less on the multi-limb support time for walking stability, which is enhanced.
The present study has shown that TLJN can improve stance time and changes of duty cycle patterns in cerebral ischemia animals. This study also shows that the swing speed of the four limbs significantly decreased in the cerebral ischemia animals in the swing phase and that TLJN can significantly improve the moving speed of the limbs during walking by increasing the swing speed of the limbs in these animals. This shows that TLJN not only improves the gait parameters in the stance phase but also increases the gait efficiency of the four limbs in the swing phase, which helps achieve the increase in overall locomotive speed.
Limb coordination is a key characteristic of movement; thus, parameters related to limb coordination are of great significance. Tetrapods’ ability to walk primarily relies on coordination provided by the nervous system in the spinal cord, which produces a basic form of movement. These types of nervous system networks are collectively known as central pattern generators9,10. This locomotor activity characterized by the rhythmic changes in flexors and extensors can most probably only be initiated and stopped under advanced motor center control. The complex changes in coordination may be assessed by parameters such as homology, contralateral, and ipsilateral coordination11,12. A recent study13 reported abnormalities in contralateral and homologous hindlimb coordination in an animal model of stroke with reperfusion. Wang et al.14 discovered that the homologous coordination of the ipsilateral hindlimbs relative to the contralateral hindlimbs was significantly worse in animals with cerebral ischemia than in sham surgery rats. One important point to consider is the effect of speed on coordination parameters and that the conversion from walking to trotting will affect contralateral coordination to some extent. Therefore, the comparison of contralateral and ipsilateral coordination in tetrapods is only meaningful when the speed is similar. Nonetheless, for symmetrical gaits such as walking and trotting, homologous coordination should not be affected by speed11. In this study, the speed of the pMCAO rats was significantly slower than that of the sham surgery rats. Therefore, our primary observation was the significant changes in the homologous coordination of pMCAO rats. Additionally, movement coordination disorders may also be an important reason for the decrease in locomotor speed of the cerebral ischemic animals. TLJN can significantly improve the coordination disorders in the animals, which may be a pharmacodynamic basis of its effects on increasing locomotor speed of the cerebral ischemic animals.
In conclusion, this study demonstrated that TLJN significantly improved the cognitive and gait deficits of rats with cerebral ischemia, and its mid- to long-term efficacy was mainly reflected 4 weeks after drug treatment. The results also showed that the behavioral outcomes of this study are consistent with the pathological findings. Interestingly, 2 weeks after TLJN administration to the cerebral ischemic rats, their walking speed was significantly increased. Although improvement trends were noted in some gait parameters 2 weeks after treatment, most parameters did not display significant differences. It is speculated that the pharmacological effects of TLJN 2 weeks after treatment in cerebral ischemic rats have not yet exerted significant differences in every movement aspect of their gait. Nonetheless, the multiple improvement trends shown in most movement aspects affect walking speed, which can explain the significant difference in this gait parameter that reflects overall gait function. In other words, minor and insignificant changes 2 weeks after TLJN treatment in cerebral ischemic animals together lead to a large and significant improvement in walking speed.
National Major New Drug Development Program (2015ZX09501004-003-002);A project of the International S&T Cooperation Program of China, 2011DFA32730.
The authors declare that they have no competing interests.
- 1. Zou XJ;Lai LH;Jin GY, et al. Studies on the 3D-QSAR of Novel 1-aryl-1,4-dihydro-3-acylhydrazinocarbonyl-6-methyl- 4-pyridazinones. Acta Physico-Chimica Sinica 2002;18(6): 513-516.
- 2. Gu Y, Chen MB,Dong XC, et al.3D - QSAR Studies of Saponins.Acta Chimica Sinica. 2000, 58(12): 1534-1539.
- 3. XU Man,ZHANG Ai-qian,HAN Shuo-kui,WANG Lian-sheng,et al.Study Progress on 3D-QSAR.Reserrch of Environmental Science,2002,15(1):45-47.
- 4. Merino JG, Hachinski V. Stroke-related dementia. Curr Atheroscler, 2002, Rep 4: 285–90.
- 5. Sachdev PS, Brodaty H, Valenzuela MJ, Lorentz LM, Koschera A. Progression of cognitive impairment in stroke patients. Neurology, 2004, 63: 1618–23.
- 6. Hall AL, Peterson CL, Kautz SA, Neptune RR. Relationships between muscle con-tributions to walking subtasks and functional walking status in persons with post-stroke hemiparesis. Clin Biomech (Bristol, Avon), 2011, 26: 509-15.
- 7. a. b. Nadeau S, Arsenault AB, Gravw ID, et al. Analysis of the clinical factors determining natural and maximal gait speeds in adults with a stroke [J]. Am J Phys Med Rehabil, 1999, 78(2): 123-30.
- 8. Damiano DL, Abel MF. Functional outcomes of strength training in spastic cerebral palsy [J]. Arch Phys Med Rehabil, 1998, 79: 119–125.
- 9. Cazalets JR, Borde M, Clarac F. Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat [J]. J Neurosci, 1995, 15: 4943–51.
- 10. Kjaerulff O, Kiehn O. Distribution of networks generating and coordinating locomotor activity in the neonatal rat spinal cord in vitro: a lesion study [J]. J Neurosci, 1996, 16: 5777–94.
- 11. a. b. Hamers FP, Koopmans GC, Joosten EA. CatWalk-assisted gait analysis in the assessment of spinal cord injury [J]. J Neurotrauma, 2006, 23: 537–48.
- 12. Koopmans GC, Brans M, Gomez-Pinilla F, Duis S, Gispen WH, Torres-Aleman I, Joosten EA, Hamers FP. Circulating insulin-like growth factor I and functional recovery from spinal cord injury under enriched housing conditions [J]. Eur J Neurosci, 2006, 23: 1035–46.
- 13. Hetze S, Römer C, Teufelhart C, Meisel A, Engel O. Gait analysis as a method for assessing neurological outcome in a mouse model of stroke [J]. Journal of Neuroscience Methods, 2012, 206: 7–14.
- 14. Wang Y, Bontempi B, Hong SM, Mehta K, Weinstein PR, Abrams GM, et al. A comprehensive analysis of gait impairment after experimental stroke and the therapeutic effect of environmental enrichment in rats [J]. J Cereb Blood Flow Metab, 2008, 28: 1936–50.
目的：探讨通络救脑口服液（TLJN Oral Solution）对局灶性脑缺血模型大鼠认知和步行功能障碍的改善作用。