动物模型,脊髓损伤 z-bo 2020-07-23 668

　　 In 1953, Tarlov and colleagues described the open field experiment for the first time and used it to evaluate the motor function of animals after spinal cord compression injury. The content includes joint mobility, walking or running. Its characteristics are more reliable for primates, and have a better correlation with the degree of spinal cord injury, nerve function recovery and the number of residual axons, but it is less consistent with rodents. I will. Due to the subjective arbitrariness of the observer, the reproducibility is not high in different experimental environments. Then, many scholars made many improvements to the Tarlov method and used it to evaluate hindlimb function in rats. Kazanci et al. used the Tarlov method to evaluate the motor function after spinal cord ischemic injury in rabbits, which showed that mexiletine has an important role in improving nerve function and reducing histopathological damage. However, there is no statistical difference compared with methylprednisolone (a powerful antioxidant). Liang et al. studied the combined effect of gustazine and deferoxamine on spinal cord ischemic injury in rats and found that the Tallov score of the treatment group was significantly higher than that of the control group, which was related to the incidence and morphological changes of paraplegia. I found out. The improved Tarlov method is relatively simple, but the score range is large and easy to jump distribution. It is difficult to distinguish between different degrees of rodent SCI, and it is also difficult to reveal the entire process of nervous system 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 grading standard and tilted plate test to evaluate the improvement of hind limb function in SCI rats treated with SJA6017 (calpain inhibitor), which may be due to the inhibition of calpain-induced apoptosis. SCI treatment strategy. 2. Basso method (Basso-Beattie-Bresnahantest) In 1995, Basso et al. proposed a new neuromotor function assessment method (referred to as the BBB method) based on the field test of the Tarlov method. The classification of this method is more detailed, including almost all behavior changes during the recovery of the hind limbs of rats after spinal cord injury, and is very consistent with the degree of spinal cord injury. Currently, many researchers recommend this method. The most important thing is to put the animal in a closed circular metal shell, and two observers stand on opposite sides to observe the changes in the movement function of the hind limbs. The observation period is 5 minutes, during which the observer 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 has been used to evaluate the recovery of hindlimb motor function in rats after SCI, especially the motor function after lower thoracic spinal cord contusion. Second, the scoring results are flexible and can be used to explore the mechanism of motor function recovery. The scores are graded so that they reflect early, mid, and late behavior changes, thereby revealing the entire process of SCI recovery. Based on this, Lankhorst et al. added data input, storage and analysis functions to facilitate comparison between data. Li et al. improved the BBB method and applied it to the evaluation of motor function in a mouse SCI model. It has higher sensitivity than the inclined plate test, and the coefficient of variation of this method is very small. 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, which requires further research. Takeda et al. reported that minocycline was used to treat ischemic spinal cord injury in rats, and then the BBB method was used to evaluate the motor function of the hind limbs of rats. 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 using the BBB method to evaluate the motor function of the forelimbs and hindlimbs, two double-blind independent observations and records should be used to make the score more objective. For beginners, many scoring details are difficult to understand. The BBB method is only suitable for mild and moderate injuries, but it is not very sensitive to serious injuries.

3. Gait analysis

　　DeMedinaceli et al. established the footprint analysis method in 1982 and applied it to the evaluation of rat neuromotor function, and then Mets et al. made many improvements. Different colors of ink were applied to the legs of the experimental animals to walk on wooden sticks, and the number of steps, the load of the legs and the angle of movement of the legs were used as indicators for analysis. However, it is difficult and accurate to accurately measure the step distance of the animal and the movement angle of the nail. Jia et al. According to reports, the traditional Chinese medicine Shuxuetong can significantly promote blood circulation in the spinal cord, reduce secondary damage, and protect neurons in ischemic areas. Using Tarlov score and footprint analysis, we determined that Shuxuetong can promote the recovery of motor function of injured spinal cord. Following the BBB method, the VisuGait analysis system is an automatic evaluation system for studying animal gait, which is widely respected in the academic world. We recommend using the VisuGait analysis system to confirm the results of the main proprietary experimental therapies. This method can provide a large amount of analysis data for various motor functions, such as landing time, suspension time, stride length, distance between left and right feet, and step sequence. Compared with the BBB method, the VisuGait analysis system overcomes the shortcomings that it is difficult to make accurate assessments due to the rapid movement of animals, and is suitable for the assessment of fore and hind limb coordination. Secondly, this method also greatly reduces the influence of human factors and greatly improves the reliability of the evaluation results. Salazar et al. used VisuGait analysis system and BBB method to observe the therapeutic effect of human neural stem cell transplantation 30 days after spinal cord contusion in mice. The results showed that the motor function of mice was significantly improved on the second day after transplantation.

　　Treadmill test is another improvement based on VisuGait analysis system. The main purpose is to convert the transparent track into a running belt to facilitate gait analysis. This test can be used for the mechanical analysis of animal limb coordination and horizontal or diagonal gait, and is suitable for the study of small rodents. Secondly, the gait parameters are related to the speed of the running belt, which helps to evaluate the neuromotor function at various stages. This method eliminates the need for ink on the forelimbs and hind limbs of the tested animal, thereby making up for the flaws in the footprint analysis. Therefore, although the treadmill test has been widely used to study the sensory motor cortex damage and nerve regeneration in the brain after SCI, it is still necessary to purchase an expensive treadmill equipped with a high-speed camera and supporting data acquisition and analysis software. There is a downside. Li et al. reported that adult central nervous system injury, treadmill test, modified, modified BBB method, inclined board test, activation of Nogo-66 receptor may promote axon growth. All graded grid crawl scores have been significantly improved.

　　4. Grid walking

　　 Grid walking test is an evaluation method that can detect whether an animal has precise control over the position of the hindfoot after brain or spinal cord injury. 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. In this method, the test animal is placed on a horizontal or inclined grid (the distance between the two rods is 2.5 cm), the animal is trained to find food and water on the grid, and the rats are fed in the process . Record the number of times the hind legs are taken off. Behavioral data, such as the number of footsteps created and the time to travel this distance. The disadvantage is that the grid lines are too thin to evaluate the hind limbs. The trainer needs to know all the details of the assessment in order to accurately distinguish between injured and uninjured behavior changes. Human factors have high influence, time-consuming, labor-intensive and high-cost. It is not easy to observe animals walking too fast. Later, Prakriya and others established an automated analysis system for grid crawl testing. It was originally used to evaluate the behavioral changes after T12-T13 spinal cord transection injury. Behavioral decisions, especially the continuity of steps, are more accurate. It has the advantages of being easy to operate, time-consuming and highly comparable. However, subtle changes in behavior (such as slight slips) are more difficult to detect. In addition, due to the limitations of the one-dimensional sensory model, it is difficult to distinguish the footprints of the left and right hind limbs. Xu et al. combined neural stem cell transplantation with NgR vaccination to improve the motor function of adult rats after SCI. The grid crawl test index and BBB score are far superior to the therapeutic effect of a single drug.

　　5. Balance beam walking

　　 Balance beam walking can be divided into two types of tests: trapezoidal beam test and narrow beam test. The former is used to assess the animal's ability to control the placement of the front and hind paws. The method involves placing the animal on the foot balance bar, recording the number of times the front feet and hind paws fall off the balance bar, and collecting data from DV video. Cummings et al. created an improved stepped beam device and its scoring system, and used it to evaluate the hindlimb function of mice with T9 spinal cord contusion. Most importantly, the horizontal walking test can distinguish mice with the same footprint in the field test. The characteristics of this method are that the training time of the subjects is short, the parameter evaluation is accurate, and the test process is convenient for repeated observation. Then, it helps to distinguish behavior changes between mouse BBB scores 5–7 and rats BBB scores 9–13. However, unlike the BBB method, this method mainly evaluates sensorimotor cortex injury or dysfunction of the forelimbs and hindlimbs caused by SCI (especially high neck SCI). In addition, this method can distinguish the functional recovery capacity of certain areas, such as weight when walking. Fiore et al. tilted the ladder equipment to 40° and established a ladder-type balance beam test to analyze missed mouse pointers. After treating moderate spinal cord contusions in mice with the neurosteroid dehydroepiandrosterone, motor behavior deficits were reduced, and behavioral outcomes were related to neuropathological changes. In 1975, Hicks and others established a slender average beam device to evaluate the balance ability of rats after SCI, the number of paws missed, and other indicators. It is suitable for SCI model and behavioral evaluation of skull sensorimotor cortex injury. According to the test requirements, the balance beam is usually divided into three specifications. Kunkel-Bagden et al. believe that the narrow balance beam test can quantitatively evaluate the motor function of SCI rats. Guo Liping and colleagues used a modified stenosis balance beam test to evaluate the ability of rats to coordinate muscle movement and maintain balance after brain injury, thereby quantifying the degree of injury. The disadvantage is that the narrower the balance beam, the more steps the experimental animals miss, and the worse the reliability of the evaluation results. Merkler et al. found significant results in the study of the inhibitory effect of Nogo-A (myelin-associated axon growth inhibitor) antibody on neutralizing inhibitory antigens, narrow balance beam test, BBB score and crawling grid test score. It has been reported to have improved.
6. Thoracolumbarheighttest Thoracolumbarheighttest is a device that monitors the chest and waist height through a device. When a mouse passes through the transparent track corridor, the device can monitor the chest and waist height. Observe and analyze whether the hind limbs of the rat are partially or fully loaded. VandeMeent et al. The rats were placed in three different types of environments after SCI, and the behavioral changes were evaluated through the chest-waist height test, BBB score, VisuGait gait analysis and grid test. The results show that when training SCI rats reach certain thresholds in a rich environment, the motor function will be significantly improved, but the recovery of motor function will not increase with the increase of intensity. I will. The disadvantage is that it is not suitable for behavioral evaluation of lightweight or very heavy SCI models, and can only be used as an auxiliary tool for behavioral evaluation.

　　7. Tilted plane test

　　 Inclined plane test is mainly composed of two right-angle splints interconnected by hinges. There is a corner plate on the side of the inclined plate, which can easily adjust the angle. The method is to put the experimental animal on the inclined plate and adjust the angle of the inclined plate to obtain the animal's SCI, and then obtain the maximum angle value of 5 seconds. The inclined board test equipment is simple, easy to use, repeatable, non-invasive, highly correlated with the degree of SCI, and suitable for mild to moderate SCI models. Yonemori et al. innovated the inclined plate test in 1998. Specifically, place the rat on a horizontal swash plate (0°), gradually increase the starting angle to 30°, and then increase the animal at a speed of 2°/sec until it slides out of the swash plate and reaches the maximum angle Value. recording. .. Han et al. reported that after taking the lipid-lowering drug simvastatin in experimental SCI rats, the angle of the inclined plate test and the BBB score were significantly improved. It is believed that this neuroprotective effect is related to the up-regulation of BDNF and GDNF expression. Domestic scholars have observed the recovery of hindlimb function in rats after spinal cord injury through the combination of the tilt-plate test and the Tarlov method. In order to improve the reliability of the method, the entire experimental process is usually divided into two stages: the preliminary stage and the formal stage. The purpose of the preliminary experiment is to adapt the experimental animals to the environment and avoid fear that will affect the evaluation results in the formal experiment. The disadvantage of the swash plate test is that it is difficult to reveal subtle changes in rat nerve function, such as paw position, sagging or upward bending, and affect the overall assessment of motor function. Song Huanjin and colleagues observed the recovery of lower limb motor function after SCI-treated rats Ginkgo biloba extract, and found that the values detected by the swash plate test and BBB method were significantly related to the degree of spinal cord injury. Find. It is believed that the 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 scoring.

　　 8. Limb muscle strength test (limbmusclestrengthtest)

　　 Limb muscle strength test can be divided into limb suspension test and limb grip test according to various experimental devices. The former can be used to evaluate forelimb function after SCI, especially forelimb muscle function. In the limb suspension test established by Diener et al., the device consisted 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 the suspended wooden stick and the ability and time of the animal to hold the wooden stick were tested to assess the strength of the limb muscles. Compared with the Tarlov method, the evaluation results of animal models with moderate to severe injuries are more accurate. The disadvantage is that it is not susceptible to mild or severe injuries and must be used in combination with other behavioral methods. The method of limb grip test is to test the animal by grasping the hoop attached to the grip meter, and use the grip meter reading to measure the recovery of the animal's motor muscle strength. Anderson et al. It is pointed out that the grip test of limbs is convenient and can be quantitatively analyzed. It is suitable for evaluating forelimb motor function in SCI cervical model, but not suitable for severe injury model. Aguilar et al. used a modified limb grip test to evaluate the forelimb motor function of C5 bilateral SCI mice. Add some indicators (such as sensation and reflex) to establish a complex scoring method (CBS) including hind limb movement, tilt board test (such as Gail based on a combination of Tarlov and tilt board method). Seven items including toe extension, contraction response, hot plate test and swimming. Each value represents the percentage of neurological loss after SCI. The CBS method provides a more accurate and comprehensive assessment of the motor nerve and sensory nerve function in SCI rats. The disadvantage is that the required equipment is more complicated, and there are many human factors that make it impossible to upgrade. Since then, Kelasidis and his colleagues have revised the CBS scoring criteria, canceling the "routine walking: 0 points" option, and said "the limbs bear weight and can walk 1-2 times". You can walk several times. ", thereby reducing human factors and improving the reliability of the score. Von Euler and colleagues confirmed that the motor function results assessed by CBS and BBB are closely related to the histological changes in different degrees of spinal cord compression injury models. Hara et al. reported. The effectiveness of fasudil hydrochloride (an isoquinoline sulfonamide derivative) in the treatment of T3 spinal cord compression injury in rats. In this study, a modified CBS was used to evaluate the motor function of SCI animals. The behavioral indicators of the methylprednisolone group were significantly better than those of the methylprednisolone control group. Domestic scholars are studying the effects of surgical decompression on chronic spinal cord injury in rats and related mechanisms. CBS results show that surgical decompression can enhance spinal cord motor neurons. Acetylcholine transferase is synthesized and improves the motor function of animals.