脊髓损伤动物模型 z-bo 2020-12-09 366

　　Abstract: Different animal models of spinal cord injury replicate different types of anatomical structure injuries and corresponding behavioral changes. Motor function evaluation after injury can not only directly measure the regeneration and repair of the spinal cord, and the reconstruction of nerve function, but also an indispensable link in the development of neuroprotective drug pharmacodynamic tests. This article makes a systematic review of the evaluation methods that have been established at home and abroad, and at the same time enumerates their applications in experimental therapeutics, and guides researchers to choose appropriate evaluation methods. Spinal cord injury (SCI) is due to the displacement of the vertebral body or the protruding of bone fragments in the spinal canal, which causes varying degrees of damage to the spinal cord or cauda equina, and various motor, sensory and sphincter functions corresponding to the injured segment Disorders, abnormal muscle tone, and pathological reflexes. Various animal models of SCI such as spinal cord contusion, compression injury, transection injury, ischemia injury, stretch injury and chemical injury have been established at home and abroad, and pharmacodynamic screening of some neuroprotective drugs has been carried out. However, in actual research, it is found that there is still a big difference between animal models and clinical SCI. It is necessary to establish a complete and objective motor function evaluation system. This is particularly important for further exploring the mechanism of SCI occurrence and development and repair treatment after injury. Existing SCI animal models of motor function evaluation methods can be roughly divided into open field test (open field test), non-open field test (non-open field test) and combined behavioral score method (combined be-havioral score) 3 class. This article will systematically review the motor function evaluation methods of various SCI animal models and their advantages and disadvantages, so that researchers can choose appropriate models and evaluation methods according to the needs of experimental therapeutics.

　　1.Tarlov method (Tarlov test)

　　In 1953, Tarlov et al. described the open field test for the first time, and it was used to evaluate the motor function of animals after spinal cord compression injury. The content included joint mobility, whether they could walk or run, etc. Its characteristic is that it is more reliable for primates, and has good correlation with the degree of spinal cord injury, nerve function recovery and the number of residual axons, but it is less consistent for rodents. Due to the subjective arbitrariness of the observer, the repeatability is not high in different experimental environments. Subsequently, many scholars made many improvements to the Tar-lov method and applied this method to the evaluation of hindlimb function in rats. Kazanci et al. used the Tarlov method to evaluate the motor function after spinal cord ischemic injury in rabbits, indicating that mexiletine has a significant effect on improving nerve function and alleviating histopathological damage, but there is no statistical difference compared with methylprednisolone (a powerful antioxidant) . Liang et al. explored the combined effect of ligustrazine and deferoxamine in the treatment of spinal cord ischemic injury in rats, and found that the Tarlov score of the treatment group was significantly higher than that of the control group, and the incidence of paraplegia was correlated with morphological changes. Although the modified Tarlov method is relatively simple, it has a large scoring span and is prone to jumping distribution. It is difficult to distinguish the difference in the degree of SCI in rodents, and it is also difficult to reveal the entire process of neurological recovery. Therefore, the Tarlov method is only used as a preliminary screening of the degree of SCI in rodents, and is suitable for use in combination with other behavioral methods. Akdemir et al. used the modified Tarlov method grading standard and inclined plate test to evaluate the improvement of hindlimb function in SCI rats after treatment with SJA6017 (a calpain inhibitor), suggesting that inhibition of calpain-induced apoptosis may be a factor. A feasible SCI treatment strategy.

　　2. BBB method (Basso-Beattie-Bresnahan test)

　　In 1995, Basso et al. proposed a new neuromotor function evaluation method (abbreviated as BBB method) based on the open field test of Tarlov method. The classification of this method is more detailed, including almost all behavioral changes during the recovery of the hind limbs of rats after SCI, and it is highly consistent with the degree of spinal cord injury. This method is currently a method that many researchers recommend. The main purpose is to place the animal in a circular closed metal shell. Two observers stand on opposite sides to observe the movement function of the hind limbs. The observation period is 5 minutes, during which the observers will score according to the scoring standard. According to the scoring results, SCI can be divided into four stages: paralysis, early recovery, mid-term recovery and final recovery. Basso et al. believe that this method is specifically used to evaluate the recovery of hindlimb motor function in rats after SCI, especially the motor function evaluation after low thoracic spinal cord contusion. Second, the scoring results are flexible and can be used to explore the mechanism of motor function recovery. As the scores are arranged progressively, they can reflect early, mid and late behavior changes and reveal the entire process of SCI recovery. Lankhorst et al. added data input, storage, and analysis on this basis, facilitating data comparison. Li et al. improved the BBB method and applied it to the motor function evaluation of the mouse SCI model. Compared with the inclined plate test, it is more sensitive, and the coefficient of variation of this method is smaller. Pinzon et al. reported that minocycline treatment did not cause behavioral or histopathological changes in SCI rats, and called for the use of minocycline treatment after clinical spinal cord contusion that requires further detailed research. Takeda et al. reported the use of minocycline to treat spinal cord ischemic injury in rats, and then used the BBB method to evaluate the motor function of the rat's hind limbs. The results showed that the BBB score and the number of normal neurons in the treatment group were significantly higher than those in the control group. When the BBB method is used to evaluate the motor function of the front and rear limbs, it is necessary to use double-blind, two-person independent observation records, so that the score results are more objective. It is not easy to grasp for beginners due to the many details of scoring. The BBB method is only suitable for light and moderate injuries, and the judgment of heavy injuries is not sensitive.

　　3. gait analysis

　　De Medinaceli et al. first established a foot-print analysis method in 1982, and applied it to the evaluation of neuromotor function in rats, which was later improved by Metz et al. In this method, the paws of test animals are painted with ink of different colors and allowed to walk on wooden poles. The analysis is carried out with the index of stepping distance, paw load and paw movement angle. However, it is difficult to accurately measure the animal's stepping distance and the angle of movement of the paw, and the reliability is poor. Jia et al. reported that Shuxuetong, a traditional Chinese medicine, can significantly promote spinal cord blood circulation, reduce secondary damage, and protect neurons in ischemic areas. Tarlov scoring method and footprint analysis method were used to prove that Shuxuetong can promote the recovery of motor function of injured spinal cord. After the BBB method, the VisuGait analysis system is an automatic evaluation system for studying animal gait that is widely respected in the academic circles. It is recommended that the VisuGait analysis system should be used to verify the results of major and original experimental therapeutics. This method can provide a large number of analysis data of different motion functions, including landing time, hanging time, step length, left and right foot spacing, step sequence, etc. Compared with the BBB method, the VisuGait analysis system overcomes the defect that it is difficult to make an accurate assessment due to the rapid movement of the animal, and is suitable for the evaluation of fore-and-fore-limb coordination. Secondly, the method also greatly reduces the influence of human factors, which greatly improves the reliability of the evaluation results. Salazar et al. used VisuGait analysis system and BBB method to observe the therapeutic effect of transplantation of human neural stem cells 30 days after spinal cord contusion in mice. The results showed that the motor function of mice was significantly improved on d 2 after transplantation.

　　treadmill test is another improvement based on the VisuGait analysis system. The main purpose is to convert the transparent track into a running running belt, which is more conducive to gait analysis. This test can be used for the analysis of animal limb coordination and horizontal or inclined gait mechanics, and is suitable for the study of small rodents. Second, the gait parameters are correlated with the speed of the running belt, which facilitates the assessment of neuromotor function at different stages. This method eliminates the need to apply ink to the fore and hind limbs of the test animal, which makes up for the defect of footprint analysis. Therefore, the treadmill test is widely used in the study of brain sensorimotor cortex injury and nerve regeneration after SCI. Its disadvantage is the need to purchase expensive treadmills with high-speed cameras and supporting data acquisition and analysis software. Li et al. reported that activation of Nogo-66 receptors can promote axon growth after adult central nervous system injury, and the treadmill test, modified, modified BBB method, inclined board test, and inclined grid crawl scores have all been significantly improved.

　　4. Grid walking

　　Grid-climbing test is an evaluation method to detect whether animals have the ability to accurately control the placement of their hind paws after brain or spinal cord injury. It is suitable for small animals such as cats and mice.

　　According to different grid placement angles, it can be divided into horizontal grid test and inclined grid test. The method is to place the test animal on a horizontal or inclined grid (the distance between the two rods is 2.5 cm) to train the animal to look for food and water above the grid, and record that the rat's hind paws missed during this process Behavioral data such as the number of times, the number of footsteps made and the time to pass this distance. The disadvantage is that the grid lines are too thin, making it difficult to evaluate hind limbs. The trainer is required to grasp every detail of the evaluation in order to accurately distinguish the injury and non-injury behavior changes. Human factors have a greater impact, time-consuming, labor-intensive, and costly. It is not easy to observe when animals are walking too fast. Later, Prakriya et al. established an automatic analysis system for the grid crawl test, which was first used to evaluate the behavioral changes after the T12-T13 spinal cord transection injury. The behavioral judgment is more precise, especially the judgment of the continuity of footsteps. It has the advantages of simple operation, short time-consuming and high comparability. However, small changes in behavior, such as minor slips, are still more difficult to detect. In addition, due to the limitations of the one-dimensional sensory model, it is difficult to distinguish the footsteps of the left and right hind limbs. Xu et al. combined neural stem cell transplantation and NgR vaccination to improve the motor function of adult rats after SCI. The grid crawl test index and BBB score were significantly better than the treatment effect of a single drug.

　　5. Walking on a balance beam

　　balance beam walking can be divided into two types of tests: ladder beam test and narrow beam test. The former is used to evaluate the animal's ability to control the placement of the front and rear paws. The method is to place the animal on a step balance lever, record the number of times the front and rear limbs fall off the lever, and collect data from DV video. Cummings et al. produced an improved stepped balance beam device and its scoring system, and used it to evaluate the hindlimb function of mice with T9 spinal cord contusion. The most important thing is that the horizontal stepped balance beam walking test can distinguish mice with equal footsteps in the open field test. The characteristics of this method are that the subject has a short training time, accurate parameter evaluation, and the test process is convenient for repeated observation. Secondly, it helps to distinguish the behavioral changes between points 5-7 in the BBB score of mice and points 9-13 in the BBB score of rats. But different from the BBB method, this method mainly evaluates sensorimotor cortex injury or SCI, especially fore and hind limb dysfunction caused by high neck SCI. In addition, this method can distinguish the functional recovery ability of certain areas, such as walking with weight. Fiore et al. tilted the ladder device to 40° to establish a tilted ladder balance beam test to analyze the missed indicators of mice. After moderate spinal cord contusion in mice was treated with the neurosteroid dehydroepiandrosterone, the motor behavior disorder was reduced, and the behavioral results were correlated with neuropathological changes. In 1975, Hicks and others established a long and narrow balance beam device to evaluate the balance ability of rats after SCI, the number of missed feet and other indicators, which is suitable for SCI model and behavioral evaluation of brain sensorimotor cortex injury. According to the test requirements, the balance beam is usually divided into three specifications: 1. 2 cm, 2. 3 cm rectangular balance beam and 2.5 cm diameter cylindrical balance beam. Kunkel-Bagden et al. believe that the narrow balance beam test can quantitatively evaluate the motor function of SCI rats. Guo Liping and others used a modified stenosis balance beam test to evaluate the ability of coordinating muscle movement and maintaining balance after brain injury in rats, and quantify the degree of damage. The disadvantage is that the narrower the balance beam, the greater the number of missed steps of the experimental animals, which reduces the reliability of the evaluation results. Merkler et al. reported that in the study of the inhibitory effect of Nogo-A (a myelin-related axon growth inhibitor) antibody to neutralize inhibitory antigens, the results of the stenosis balance beam test, the BBB score and the crawling grid test score were significantly improved.

　　6. thoracolumbar height test

　　Chest and waist height test is to observe the height of the thoracic and lumbar spine when the rat passes through the transparent track corridor through the monitoring device to analyze whether the rat's hind limbs are partly or fully loaded. Van de Meent et al. put the rats after SCI in three different types of environments, and evaluated their behavioral changes by chest-waist height test, BBB score, VisuGait gait analysis, and grid test. The results show that when SCI rats train in an affluent environment to reach a certain threshold, their motor function will be significantly improved, but the recovery of motor function will no longer increase with increasing intensity. The disadvantage is that it is not suitable for behavioral evaluation of light or very heavy SCI models, and can only be used as an auxiliary means of behavioral evaluation.

　　7. Inclined plane test The

　　inclined plate test device is mainly composed of two right-angle splints, which are connected to each other by a hinge. There is an angle plate on the side of the inclined plate for easy angle adjustment. The method is to place the experimental animal on an inclined plate, and obtain the maximum angle value of 5 s after the animal SCI is obtained by adjusting the angle of the inclined plate. The equipment of the inclined plate test is simple to make, the method is simple, the method is good, it is non-invasive, and has a high correlation with the degree of SCI. It is more suitable for mild to moderate SCI models. In 1998, Yonemori et al. technically modified the inclined plate test. Specifically, the rat is placed on a horizontal inclined board (0°), and then gradually rises to 30° as the starting angle, and then increases at a speed of 2 degrees/sec until the animal slides off the inclined board, and the maximum angle value is recorded . Han et al. reported that after experimental SCI rats were given the lipid-lowering drug simvastatin, the angle of the inclined plate test and the BBB score were significantly improved. They believed that this neuroprotective effect may be related to the upregulation of the expression of BDNF and GDNF. Domestic scholars have used the method of combining the inclined plate test and the Tarlov method to observe the effect of tortoise shell on the hind limbs of rats with SCI

　　Functional recovery. In order to make the method more reliable, the whole experiment process is usually divided into two stages: pre-experiment and formal experiment. The purpose of the pre-experiment is to adapt the experimental animals to the environment and avoid the appearance of fear in the formal experiment that would affect the evaluation results. The disadvantage of the oblique plate test is that it is difficult to reveal subtle changes in the rat's nerve function, such as the position of the paw, the drooping or upturning of the tail, which affects the overall evaluation of motor function. Song Huanjin et al. observed the recovery of lower limb motor function after Ginkgo biloba extract treatment of rats with SCI, and the results of the oblique plate test and BBB method were significantly correlated with the degree of spinal cord injury. It is believed that a combination of multiple behavioral evaluation methods can make up for the shortcomings of a single evaluation method, and effectively improve the accuracy and sensitivity of the score.

　　8. Limb muscle strength test (limb muscle strength test)

　　Limb muscle strength test can be divided into limb hanging test and limb grip strength test according to different experimental devices. The former can be used for the evaluation of forelimb function, especially the forelimb muscle function evaluation after cervical SCI. In the limb suspension test established by Diener et al., the device consists of a wooden rod with a length of 15 cm and a diameter of 2 mm. During the test, the animal’s front paws were gently placed on a hanging wooden stick, and the limb muscle strength was evaluated by testing the animal’s ability and time to hold the wooden stick. Compared with the Tarlov method, the evaluation results are more accurate for animal models with moderate to severe injuries. The disadvantage is that it has low sensitivity to mild or very severe injuries and must be used in combination with other behavioral methods. The method of limb grip strength test is to test the animal by gripping the ferrule attached to the grip strength meter, and use the reading in the grip strength meter to measure the recovery of the animal's exercise muscle strength. Anderson et al. pointed out that the limb grip test is convenient and can be quantitatively analyzed. It is suitable for the evaluation of the forelimb motor function of the cervical SCI model, but it is not suitable for the severe injury model. Aguilar et al. used a modified limb grip test to evaluate the motor function of the forelimbs after bilateral SCI in mice C5.

　　9. Combined behavioral score (combined behavioral score) Gale et al. established a joint scoring method (CBS) based on the combination of the Tarlov method and the inclined board method by adding some indicators such as sensation and reflex. The content includes hind limb movement, inclined board test, toe extension, retraction response, hot board test and swimming Wait 7 items. Each value represents the percentage of neurological loss after SCI. The CBS method can comprehensively evaluate the motor and sensory nerve functions of SCI rats more accurately. The disadvantage is that the required equipment is more complicated and there are many human factors that are not conducive to promotion. After that, Kerasidis et al. revised the CBS scoring standard by deleting the "normal walking: 0 points" option, and changing "the hind limbs can bear weight and can walk 1 or 2 times" to "the hind limbs can bear weight and can walk several times" , Thereby reducing human factors and improving the reliability of scoring. von Euler et al. confirmed on different degrees of spinal cord compression injury models that the results of motor function evaluated by CBS method and BBB method have a good correlation with histological changes. Hara et al. reported the efficacy of fasudil hydrochloride (isoquinoline sulfonamide derivative) in the treatment of rat T3 spinal cord compression injury. In the study, a modified CBS was used to evaluate the motor function of SCI animals. As a result, the behavioral indicators of the drug group were significantly better than those of the methylprednisolone control group. Domestic scholars have studied the effects of surgical decompression on chronic SCI in rats and related mechanisms. CBS results show that surgical decompression therapy can promote the synthesis of acetylcholine transferase by spinal cord motor neurons and improve animal motor function.

　　Clinical SCI often involves multiple types of injuries coexisting with each other, and a single type of injury is rare. Different animal models of spinal cord injury replicate different Chinese Pharmacological Bulletin 2011 Jul; 27( 7) ·895· Types of anatomical structure damage and corresponding behavioral changes, so that any animal model cannot be completely simulated Features of clinical SCI. At present, the evaluation of clinical SCI neuromotor function still follows the American Spinal Cord Injury Association/International Spinal Cord Association grading standards. The content includes three parts: evaluation of nervous system examination, evaluation of auxiliary examination (referring to imaging and electrophysiological examination) and evaluation of daily living ability. The necessary parts of the neurological examination include nerve injury level, sensory level, motor level, injury degree score, sacral remnant, partial reserved area, complete or incomplete injury, and quadriplegia or paraplegia. However, this set of grading standards still has limitations, such as functional independence assessment cannot fully reflect the recovery of injured patients. Chafetz et al. reported how to improve the accuracy of the motor sensory score, pointing out that regardless of the doctor's experience in the field of SCI, it is essential to receive formal standardized training. Therefore, the ideal animal model motor function evaluation method must be closely integrated with the actual clinical SCI evaluation criteria. First, the individual subject has little influence and can better make accurate judgments on neurobehavioral changes at different stages; secondly, it requires low cost evaluation devices. It is easy to use and the experiment takes a short time.