动物造模,打击模型 z-bo 2022-02-02 1511

　　1. The purpose of the experiment

With the development of the transportation and construction industry, the emergence of accidents and natural disasters, the incidence of traumatic brain injury (TBI) is gradually increasing. Traumatic brain injury ranks first in the global cause of death and disability due to accidental injuries. Many economic, medical and other social issues. Secondary brain injury after trauma is an important cause of death and disability in patients with craniocerebral injury. To understand the mechanism of brain injury and the behavioral changes after injury, and to formulate reasonable and effective treatment and rehabilitation measures on this basis has become the goal of clinicians. This experiment refers to the Feeney free-fall model design and appropriately improves the striking device, makes a mouse traumatic brain injury model with a certain degree of striking force, and discusses the method of making the model, the behavioral changes after injury, and its application in the study of traumatic brain injury. value.

　　 2. Experimental principle

　　 By adjusting the mass of the impact hammer and the height of its fall, it replicates different degrees of traumatic brain injury models. In the case of mild injury, this model is similar to the concussion of traumatic brain injury but without obvious contusion or focal damage. If a more powerful blow is performed, this model can create focal injuries similar to clinically seen brain contusions.

　　 3. Experimental materials and methods

　　 (1) Experimental materials

　　1. Brain injury model device The model device consists of a base, an impact rod, a drop hammer, a weight and a peripheral catheter. The diameter of the impact rod tip is 4.5mm and the height is 2.5mm. The outer circumference of the duct is 40cm high (with graduation lines), and there is an air hole every 1cm to prevent the influence of air compression resistance in the duct when the hammer falls. The iron weight is 20g and the diameter is 15mm. By adjusting the drop height of the impact hammer, different degrees of traumatic brain injury models can be replicated.

2. Experimental animals and grouping Animal selection and grouping: 20 male, healthy, month-old (7 months), and similar weight mice [(25±5)g] 20 were randomly divided into two groups: 10 in TBI group , 10 in the sham operation group. Animals eat freely, the room temperature is 22～25℃.

　　(2) Experimental method

　　1. Method for making brain trauma model The mice were fed separately 3 days in advance. Fasting for 8h before surgery. During the experiment, 3.6% chloral hydrate was injected intraperitoneally at a dose of 1ml/100g body weight, and fixed on a wooden board in the prone position; routinely disinfected the skin, cut the center, peeled off the periosteum, and exposed the right parietal bone; used a dental drill 1.5 behind the coronal suture mm, a bone window with a diameter of 5mm was drilled 2.5mm after the midline to keep the dura mater intact; the striker was placed on the dura, and a 20g weight was used to drop it at a height of 10cm, 20cm, and 30cm. Membrane, the impact forces of the three types of injury were 100g·cm, 200g·cm, and 300g·cm, which caused mild, moderate, and severe brain damage to the right parietal lobe respectively; 40,000 U of gentamicin sulfate was instilled into the incision after the blow 4 to 5 drops, close the bone window with bone wax, and suture the scalp. Care next to a constant temperature oven. In the sham operation group, only the craniotomy window was closed with bone wax without any blow. Raise alone, pay attention to heat preservation.

Postoperative care: Keep animals in separate cages after the operation, with natural light and ventilation; frequently change clean and dry rearing cages to keep them dry; strengthen nutrition, feed eggs, sunflower seeds, and place feed and drinking water in animals And scope; closely observe the animal's mental state, diet, urination and defecation, presence of limb edema and pressure, and presence or absence of bloody secretions in the urinary system; intraperitoneal injection of penicillin 100,000 u each time, twice a day; prevention of self-harm, intestines obstruction.

2. Severity score of nerve function damage Severity score of nerve function damage is currently a more commonly used neurobehavioral evaluation method for testing motor function after traumatic brain injury. It consists of 10 different tasks and is used to evaluate the motor ability of mice after injury. , Balance ability, alertness, etc. The author made some adjustments according to the scoring method of Beni-Adani et al. (2001). The mice were trained before the brain injury model. The training lasted for 1 month, so that the score of each mouse reached 10 points. After successful model building, different assessors used blind assessment methods to perform injury on day 0, day 1, day 4, day 7, day 10, day 15, day 19, day 24, and day 28 Severity score of posterior neurological impairment. The highest score is 10 points, and the lowest score is 0 points. Scoring results: 9-10 points indicate extremely severe neurological impairment; 7-8 points indicate severe neurological impairment; 5-6 points indicate moderate neurological impairment; and less than 5 points indicate mild neurological impairment. See Table 3-1 for specific scoring indicators.

　　(3) Statistical Analysis

　　 Using the SPSS17.0 statistical software package, the measurement data results are all expressed as mean ± standard deviation (x-±s), and normality and homogeneity of variances are tested for the measured results. The t test was used for intra-group comparison, and the one-way analysis of variance was used for inter-group comparison. P<0.05 indicates that the difference is statistically significant.

　　 4. Experimental results

　　(1) Results of establishment of mouse traumatic brain injury model

The mice were fully awake 2 to 3 hours after the operation. All mice in the TBI group showed loss of movement ability of the injured hind limbs, unilateral (injured side) hemiplegia, decreased muscle tension below the injury plane, and no response to acupuncture on the injured hind limbs , And cannot walk in a straight line. It shows that the TBI model was successful.

　　 (2) The results of the neurological damage severity score (NSS) in mice after traumatic brain injury

The NSS results showed that the score results of the TBI group at each time were significantly different from that of the Sham group (P<0.05), indicating that this experiment successfully established a mouse traumatic brain injury (TBI) model, and it was a moderately severe neurological Functional impairment. The scores of the severity of neurological damage in the TBI experimental group decreased gradually over time (Table 10-2 and Figure 10-1), and after the 10th day, the NSS of the TBI mice began to be less than 5 points, which is a mild neurological function damage. It is suggested that with the passage of time after TBI, the neurological function of mice is gradually recovering. And on the 28th day, athletic ability and balance ability improved significantly. Return to near normal levels.

　　5. Result analysis

　　 This experiment is based on the brain injury model created by Feeney et al. that uses a fixed-height free fall method to strike a rat with a brain injury. A heavy object is used to directly fall from a certain height and hit the animal's head to cause a focal brain injury model. The impact site is located in the parietal lobe dominates movement and somatosensory "hind limb" area, this area is relatively flat and easy to operate. Damage to this area can cause neurological dysfunctions in the contralateral limb, such as restricted mobility and slow movement. This model has a low mortality rate, and no low brainstem and medulla oblongata injuries were found, which may be related to less brainstem compression injury in the direction of the force. The model used in this experiment imposes an impact after opening the skull window to avoid the influence of the difference in skull thickness on the experimental results. The degree of injury can be clearly graded, and the degree and scope of injury of each animal in the same degree of injury group are basically the same. The mice modeled by TBI were fully awake 2 to 3 hours after the operation, and they all showed loss of movement ability of the injured hind limbs, unilateral (injured side) hemiplegia, decreased muscle tension below the injury plane, and the injured hind limbs did not respond to acupuncture. Can not walk straight; and the scores of the TBI group at each time are significantly different compared with the Sham group (P<0.05), indicating that this experiment successfully established a mouse traumatic brain injury (TBI) model. This model is simple to operate and has good controllability. It can be quantitative and basically accord with the pathological changes and pathophysiological characteristics of clinical traumatic brain injury.

　　6. Experience and experience

In order to minimize errors during the experiment and ensure the accuracy of the experimental results, the following points should be grasped when making the model: ① The anesthesia dose should be strictly controlled during anesthesia. If the anesthesia is too deep, the injured mice will easily cause respiratory depression and die. Too shallow anesthesia affects experimental operation and controllability. ②When opening the bone window, separate the right temporal muscle slightly outward, otherwise the area is too small. It is not easy to enlarge the bone window. It is better to choose about 1.5mm behind the bregma. Too close to the medial side can easily damage the superior sagittal sinus, leading to massive hemorrhage and failure of the operation; too close to the outer side can cause uneven stress on the brain, which can easily lead to uneven brain damage, with varying degrees of damage, and easily cause encephalocele. ③When making a certain degree of damage model, pay attention to maintaining the consistency of the striking height and angle of repeated experiments. To ensure that the position and intensity of each blow are consistent. ④ Pay attention to postoperative care, heat preservation, nutrition, etc.