动物造模,脊髓损伤动物模型 z-bio 2022-02-06 1057

　　Various SCl animal models have been established at home and abroad, such as spinal cord contusion, compression injury, transection injury, ischemic injury, stretch injury and chemical injury, and the 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 SCl cases. It is necessary to establish a complete and objective motor function evaluation system, which is particularly important to further explore the occurrence and development mechanism of SCl and repair treatment after injury.

　　Some scholars divide the existing motor function evaluation methods of SCI animal models into three categories: open field test, non-open field test, and combined behavioral score. class. This book provides a systematic review of the motor function evaluation methods and their advantages and disadvantages of various SCI animal models, so that researchers can choose appropriate models and evaluation methods according to the needs of experimental therapeutics.

　　一、Tarlov method (Tarlov test)

　　The assessment of the motor ability of animals after spinal cord injury directly reflects the level of spinal cord repair and regeneration, as well as the improvement of peripheral behavioral functions. In 1953, Tarlov et al. described the open field experiment for the first time and applied it to the evaluation of motor function after spinal cord compression injury in animals. The content included joint mobility, whether to 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 randomness of the observer, the repeatability is not high in different experimental environments.

　　Many scholars subsequently made many improvements to the Tarlov method. And this method was applied 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 relieving histopathological damage, but there is no statistical difference compared with methylprednisolone (a powerful antioxidant). . Liang et al. discussed the combined effect of ligustrazine and deferoxamine on spinal cord ischemic injury in rats. Results The Tarlov score of the treatment group was significantly higher than that of the control group, and there was a correlation between the incidence of paraplegia and 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 experiment to evaluate the improvement of hindlimb function in SCI rats treated with SJA6017 (a calpain inhibitor), suggesting that inhibition of calpain-induced apoptosis may be a factor. A feasible treatment strategy for SCI.

　　Two, BBB method (BBB score)

　　Tarlov's method divides the hind limb motor function evaluation after SCI into five grades: grade 1 means no voluntary movement, limited to non-reflective movements of the hip and knee joints; grade 2 means movement of the limbs, hips, knees, and ankle joints; grade 3 means Actively support body weight and uncoordinated gait, occasionally a coordinated gait; Level 4 is a coordinated gait for the forelimbs and hindlimbs, with interdigital joint movement during walking; Level 5 is a normal gait. It is more accurate to use this method to evaluate the function of injured spinal cord, but the classification is too general and not detailed enough.

　　In 1995, Basso et al. made improvements based on the open field experiment of the Tarlov method. A new 21-level neuromotor function evaluation method, referred to as the BBB method, is proposed. The classification of this method is more detailed, including almost all the behavioral changes in the hind limb recovery process of rats after SCI, and it is highly consistent with the degree of spinal cord injury. This is a method recommended by many researchers (Table 2-1). The main thing is to place the animal on a flat and non-slip experimental platform, and two observers stand on opposite sides to observe the changes in hind limb motor function. 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. This method is specifically used to evaluate the recovery of hindlimb motor function in rats after SCI, especially the evaluation of motor function after low thoracic spinal cord contusion. Since the scoring is arranged gradually, it can reflect the behavior changes in the early, middle and late stages and reveal the whole process of SCI recovery. The scoring results are flexible and can be used to explore the mechanism of motor function recovery. Compared with the inclined plate experiment, it is more sensitive and the coefficient of variation of this method is smaller.

　　Li et al. improved the BBB method and applied it to the evaluation of motor function in a mouse SCI model. Pinzon et al. reported that treatment with minocycline did not cause behavioral or histopathological changes in SCI rats, but the use of minocycline after clinical spinal cord contusion requires further detailed research. Takeda et al. reported the use of minocycline to treat rat spinal cord ischemic injury, 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.

　　However, 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 and recording, so that the scoring results are more objective. Because of the many grading details, it is not easy for beginners to grasp. The BBB method is only suitable for mild to moderate injuries, but the sensitivity to severe injuries is not high.

　　Three, gait analysis

　　Medinaceli et al. first established a foot-print analysis method in 1982, and applied it to the evaluation of neuromotor function in rats, and then made many improvements by Metz et al. In this method, the paws of experimental animals are painted with ink of different colors and allowed to walk on wooden poles. The analysis takes the step distance, the weight of the paws and the angle of movement of the paws as indicators. However, it is difficult to accurately measure the animal's stepping distance and the angle of claw movement, 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. It is recommended to use CatWalk gait analysis to confirm the major and original experimental therapeutics results. This method can provide a large number of analysis data of different sports functions. Including landing time, hanging time, step length, left and right foot spacing, step sequence, etc. Compared with the BBB method, the CatWalk gait method overcomes the shortcomings 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 hind 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 CatWalk gait analysis method 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 2 days after transplantation.

　　The treadmill test is another improvement based on the CatWalk device. The main thing is to convert the transparent track into a running running belt, so as to be more conducive to gait analysis. According to foreign literature reports, there are two types of treadmill tests: Digit gait system and Treadscan gait system. This experiment can be used for animal limb coordination and horizontal or oblique gait mechanical analysis, and is suitable for the study of small rodents. In addition, 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 experimental animals, which makes up for the shortcomings of footprint analysis. Therefore, the treadmill experiment 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. Ji et al. reported that activation of Nogo-66 receptors can promote axon growth in adult animals after central nervous system injury, and the treadmill test, modified BBB method, inclined board test and inclined grid crawling scores have all been significantly improved.

　　4. Grid walking experiment

　　Grid crawling 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 experiment and inclined grid experiment. The method is to place the experimental animal on a horizontal or inclined grid (the distance between the two rods is 2.5cm) to train the animal to look for food and water above the grid, and record the number of times the rat’s hind paw misses during this process. Behavioral data such as 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 in this method 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 a set of automatic analysis system for grid crawling experiment, which was first used to evaluate the behavioral changes after T12～T13 spinal cord transection injury. It is more precise for behavioral judgment, especially for judging the continuity of footsteps. It has the advantages of simple operation, short time-consuming, and high comparability. However, it is still difficult to detect small changes in behavior, such as minor slips. 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 index and BBB score of the grid crawl experiment were significantly better than the therapeutic effect of a single drug.

　　5. Beam walking experiment

　　The

　　balance beam walking experiment can be divided into two kinds of experiments: ladder beam and narrow beam. 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 stepped balance lever and record the number of times the front and rear limbs fall off the lever. Collect data from DV video. Cummings et al. produced an improved stepped balance beam device and its scoring system, and used it to evaluate T. Function of hind limbs in mice with spinal cord contusion. The most important thing is that the horizontal stepped balance beam walking experiment can distinguish mice with equal footsteps in the open field experiment. The characteristics of this method are the short training time of the subjects, the accurate evaluation of parameters, the convenience of repeated observations during the experiment, and it is helpful to distinguish the behavioral changes between points 5 to 7 in the BBB score of mice and points 9 to 13 in the BBB score of rats. . But unlike the BBB method, this method mainly evaluates sensorimotor cortex injury or spinal cord injury, 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. But different from the BBB method, this method mainly evaluates sensorimotor cortex injury or spinal cord injury, especially fore and hind limb dysfunction caused by high cervical spinal cord injury. 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 experiment to analyze the mice's misstep indicators. After moderate spinal cord contusion in mice was treated with the neurosteroid dehydroepiandrosterone, the motor behavior disorder was alleviated, and the behavioral results were correlated with neuropathological changes. In 1975, Hichs et al. 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 was adapted to the SCI model and behavioral evaluation of brain sensorimotor cortex injury. According to experimental requirements, the balance beam is usually divided into three specifications: 1.2cm, 2.3cm rectangular balance beam and 2.5cm diameter cylindrical balance beam. Kunkel-Bagden et al. believe that the narrow balance beam experiment can quantitatively evaluate the motor function of SCI rats. Guo Liping et al. used a modified stenosis balance beam experiment to evaluate the ability of coordinating muscle movement and maintaining balance after brain injury in rats, and quantified 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-associated axon growth inhibitor) antibody to neutralize inhibitory antigens, the results of the narrow balance beam test, BBB score and grid crawl test score were significantly improved.

　　Six, thoracolumbar height test (thoracolumbar height test)

　　The thoracic-waist height experiment 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 partially or fully loaded. Van de Meeteren et al. put the rats after SCI in 3 different types of environments, and evaluated their behavioral changes by chest-waist height experiment, BBB score and Catwalk gait analysis, and grid crawling experiment. The results show that when SCI rats are trained in a spacious 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.

　　Seven, inclined plane test (inclined plane test)

　　The

　　inclined plate experiment device is mainly composed of two right-angle splints, which are connected to each other by hinges. 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 a sloping plate, and adjust the angle of the sloping plate to obtain the animal's SCI and maintain the maximum angle value of 5s on the sloping plate. The equipment of the inclined plate experiment is simple to make, the method is simple, the repeatability 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 experiment. 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°/s until the animal slides off the inclined board, and the maximum angle value is recorded . Han et al. believe that this neuroprotective effect may be related to the up-regulation of the expression of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF). Domestic scholars have observed the recovery of hindlimb function of rats after SCI by using the method of combining the inclined plate experiment and the Tarlov method. 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 fear in the formal experiment that would affect the evaluation results. The disadvantage of the oblique plate experiment is that it is difficult to reveal the 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 the motor function. Song Huanjin et al. observed the recovery degree of lower limb motor function after Ginkgo biloba extract treatment of rats with SCI, and found that the detection value of the oblique plate test and BBB method was significantly correlated with the degree of spinal cord injury. They considered a combination of multiple behavioral evaluation methods. It makes up for the shortcomings of a single evaluation method and effectively improves the score

　　Accuracy and sensitivity.

　　8. Limb muscle strength test

　　Limb muscle strength test can be divided into limb hanging test (limb hanging test) and limb grip strength test (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 experiment 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 experiment, the animal's front paws were gently placed on a hanging wooden stick. The muscle strength of the limbs is evaluated by testing the animal's ability and time to grip the 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 is low sensitivity to mild or very severe injuries and needs to be used in combination with other behavioral methods. The method of limb grip strength experiment is to grasp the ferrule attached to the grip strength meter by the experimental animal, 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 experiment has the characteristics of convenience and quantitative analysis, which 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 of mice C5 after SCI.

　　Nine, Gale joint scoring method (the combine behavioral score, CBS)

　　This law was originally created by Gale et al. (1985). Based on the combination of the Tarlov method and the inclined plate experiment, a joint scoring method (CBS) was established by adding some indicators such as sensation and reflection, which only included rat tail flapping and hot plate experiments. It has now been improved to include 7 items such as the classification of exercise ability in the open space, the classification of toe extension ability, the classification of touch response ability, the ability of retraction reaction, the corrective reflex, the inclined board test and the swimming test.

　　These 7 items are: ①Classification of exercise ability in an open space, that is, spread a layer of paper on a desktop, and place the mouse on it to observe the spontaneous activity within 1 min; ②Classification of toe extension ability, that is, the rat lift Start, observe the extension of the toes of the rat's legs without any influence; ③Grading of the ground-touch response ability, that is, hold the rat in one hand, rub the edge of the table with its instep or side, and observe its feet on the table. Speed and precision; ④Retraction reaction ability, that is, use two fingers to pull the hind limbs, or use acupuncture on the bottom of the hind feet or squeeze the hind feet with index fingers and thumbs to observe the speed and strength of hind limbs withdrawal; ⑤Correct reflexes, that is, turn the rat into supine Observe its righting ability; ⑥Sloping board test, that is, turn the rat's head up, down, left, and right on the sloping board, and record the maximum angle that it maintains for 5s; ⑦Swimming test, that is, observe the rat’s hind limbs in the water for 45s Activities.

　　The values of the CBS method represent the percentage of neurological loss after SCI. There are two completely opposite scoring methods in application, one is considered to be normal as 0 points, and the hind limbs are completely paralyzed as 100 points; the other is considered as normal as 100 points , Total hind limb paralysis is 0 points. Because this method integrates multiple observation indicators and methods to score the spinal cord function, it can comprehensively evaluate the animal's motor and sensory functions more accurately and relatively objectively. Although the CBS method can comprehensively evaluate the motor and sensory nerve functions of SCI rats, it is not conducive to popularization due to the complicated equipment required by the method and many human factors.

　　Since then, 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 motor function results evaluated by the CBS method and the BBB method have a good correlation with the changes in tissue morphology. Hara et al. used a modified CBS to evaluate the motor function of SCI animals, and found that the behavioral indicators of the drug group were significantly better than those of the control group injected with sodium methylprednisolone. 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 motor neurons, which can significantly improve animal motor function.

　　十、BMS

　　Mouse function scoring adopts the latest international BMS (Basso Mouse Scale) scoring method, which is formulated according to the characteristics of SCI mouse motor function changes. It has been proved to be a sensitive, reliable and effective mouse motion scoring method. In particular, its secondary scoring system takes into account the possible inconsistencies in the functions of the lower limbs after spinal cord strikes, making the BMS scoring system more complete. In this experiment, it was also found that the BMS score is simple and easy to implement, and the changes in the mouse's motor function can also be specifically reflected by the BMS score. There are significant differences in the BMS scores of mice with different spinal cord injuries. In the early stage of SCI, the decrease of BMS score is not only caused by primary and secondary death of neurons due to spinal cord damage, but also related to local hemorrhage, edema and vasospasm and other acute inflammations that cause animal pain and restrict their activities. With the gradual reduction of acute inflammation, the proliferation of glial cells and the secretion of some neurotrophic factors promote the repair and regeneration of nerves, and the BMS score increases accordingly.

　　11, SCANET evaluation system

　　Due to the small size of mice, it is difficult for researchers to accurately evaluate the motor function of mice after SCI injury, and the SCANET evaluation system solves this problem. With the establishment of standardized SCI model mice becoming more and more objective, the use of SCANET system functions is easier to obtain and reappear to assess dysfunction.

　　SCANET evaluation system is a device originally used to measure the spontaneous exercise ability of experimental animals. It consists of a Plexiglas cage with a diameter of 45cm, a frame containing an infrared sensor, a closed cage and a laptop computer. The infrared sensor is placed horizontally on the entire cage, and the change speed and acceleration per unit time can be calculated from the data recorded by the computer to get the result. From the 5min data, data such as the maximum speed and maximum acceleration in animal motion will be extracted and regarded as the best evaluation index of motor function. The data of the SCANET system showed that these parameters showed significant differences in different SCI animals, such as contusion, transection and other animals. Compared with the control animals, during the entire observation period, especially in the contusion group, they were compared with those in the contusion group during the resuscitation process. The BMS score is significantly correlated.

　　From different angles to evaluate motor function after spinal cord injury, it is advisable to objectively evaluate the functional recovery of SCI model mice. The SCANET evaluation system evaluates the maximum speed and maximum acceleration of the movement of injured mice. It has the advantages of simplicity, objectivity, and science, and can supplement other existing methods to test the deficiencies of the functional evaluation of SCI model mice.

　　12, Olby score

　　For experimental animals of different species, the evaluation system of motor function is also different. Olby scoring method is a 15-point scoring system, mainly used to determine the speed and level of recovery of motor function of dogs after SCI. Using a double-blind method, two researchers who had no knowledge of the experimental interventions independently scored the gait of the experimental dogs. The average score was scientifically calculated after 0, 2, 4, and 8 weeks, similar to the BBB score.

　　In short, 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 types of anatomical structure injuries and corresponding behavioral changes, so that no animal model can completely simulate the characteristics of clinical SCI. At present, the evaluation of clinical SCI neuromotor function still uses the American Spinal Injury Association's "International Standard for Neurological Classification of Spinal Cord Injury". 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 the functional independence assessment cannot fully reflect the recovery of injured patients. An ideal animal model motor function evaluation method must be closely integrated with the clinical actual injury evaluation criteria. First, the individual subject has little impact and can better make accurate judgments on neurobehavioral changes at different stages; secondly, the evaluation device is required to be low in cost and easy to use. , The experiment takes a short time.