动物造模,面神经损伤 z-bo 2023-02-02 550

　　Background: Functional impairment caused by facial nerve muscle disease and facial nerve injury is common and serious. Aesthetic defects also bring suffering to patients, leading to social isolation and further emotional distress. These will lead to depressive symptoms and mental health problems, thereby further aggravating their dysfunction. Several clinical factors have been identified that further affect nerve damage after peripheral nerve function is restored, including repair time, repair type and patient age. Despite advances in microsurgery techniques, functional recovery after facial nerve injury is still not ideal. Associated movement, regeneration of proximal stump axons into inappropriate distal pathways, has been considered an important factor in poor functional recovery. Previous studies have shown that electrical stimulation affects the morphological and functional characteristics of neurons, including nerve branches, the speed and direction of axon growth, rapid sprouting, and axon regeneration. Some people speculate that the mechanism of BES is to induce motor axons to preferentially re-dominate on sensory axons, thereby improving overall function. In 2000, Gordon and others studied the effect of electrical stimulation on nerve regeneration after sciatic nerve in rats. The author used fluororuby (FR) and fluorescent gold (FG) to retrogradely label sciatic nerve motor neurons, and electrical stimulation significantly accelerated axon regeneration and preferentially re-innervated the sensory branches of the motor nerve. The authors also found that short-term, one-hour stimulation is as effective as long-term stimulation that lasts from a few days to a few weeks. Since then, the concept of preferential re-domination of motor axons on sensory axons induced by short-term electrical stimulation has been extensively studied and is now well established. However, it is not clear that BES reduces the linkage effect of specific motor neuron axon networks that randomly expand to inappropriate distal motor axon branches such as the facial nerve. Recently, a research team studying peripheral nerve injury and regeneration has provided some insights on this issue. By using exogenous neurotrophic factor neutralizing antibodies, including brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), the abnormal and redundant branches of facial nerve regenerating axons can be transformed into non Appropriate access. In addition, a separate research team demonstrated that BES can regulate the expression of BDNF in motor neurons. Therefore, the possible mechanism of BES may be to reduce the abnormal branches of regenerated axons after peripheral nerve injury by regulating the expression of BDNF in motor neurons. Regarding facial nerve injury and regeneration, it may mean reducing joint movement. In 2005, Brushart et al. showed that BES can promote specific innervation of sensory pathways by cutting off sensory neurons in the dorsal root ganglion. Several studies have confirmed that BES can improve the linkage effect after facial nerve injury. The main purpose of this study is to test the hypothesis that BES reduces facial nerve damage in facial paralysis. The second purpose is to observe the effect of BES on nerve function after injury.

　　Method: Experimental design: Twenty-four rats were randomly divided into four groups, with six in each group. Groups 1 and 2 received crush injuries on the main nerve, and group 2 received BES for an additional hour. Groups 3 and 4 performed transverse injuries in the main trunk, and group 4 received BES for an additional hour. In order to explore the effect of BES on the joint movement, the upper and lower main branches of the facial nerve of all animals 3 months after injury were marked with two nerve tracers. The brainstems of all animals were sliced to identify the two main branches of motor neurons. Compare the brain stem that controls motor neuron markers. In order to evaluate the effect of BES on function, facial nerve function assessment results were collected at 2, 4 and 6 weeks after surgery. The previously validated rat facial nerve model was used.

　　Research objects: 24 female wistar rats with a weight of 200-220 grams were selected for the experiment. The other two female wistar rats served as controls. The sample size is calculated based on our previous research.

　　Facial nerve function result evaluation: This model uses head fixation device, body fixation and bilateral photoelectric sensor to detect precise whisker movement as an objective measurement method of facial nerve function. Establish an evaluation model and obtain data using the method outlined by Mendez et al.

　　Data acquisition: For each subject, the tentacles were moved by providing scented stimuli. The laser micrometer itself is connected to a 32-channel digital I/O module, which receives the digital output of the laser micrometer. The I/O module is connected to the PC through a compressed DAQ chassis. The I/O module obtains laser micrometer signals at a sampling rate of 1 kHz.

　　Surgery: All non-test animals underwent head implant surgery and facial nerve surgery by a surgeon during the same anesthesia. The other 2 and 4 groups received 1 hour BES during anesthesia after nerve injury. All rats were anesthetized with 3-4% isoflurane. Then use 1.5% isoflurane to maintain general anesthesia. Use an electric shaver to remove hair from the front of the face and top of the head.

　　Facial nerve surgery: All facial nerve surgery performed a small incision on the right side of the face of all non-control animals, just below the bony protrusion of the right ear. The parotid glands are visible under the microscope. Eversion to view the surgical field of view. The distal branch of the facial nerve is lower than the parotid bed. Determine the branches of the buccal branch and marginal mandibular branch of the facial nerve. Once determined, the proximal area of the facial nerve bifurcation was carefully dissected. The first and second groups of nerves received crush injuries. The hemostatic instrument is applied to clamp the proximal branch of the facial nerve for 30 seconds. Groups 3 and 4 received nerve transection injury. A single, sharp cross-section of the proximal bifurcated facial nerve was made with a direct microscope; the end of the nerve was cut and immediately repaired with a direct end-to-end technique. Using 9-0 sutures, make four simple interrupted sutures at the proximal and distal epineurium nerve endings. Nursing measures were taken to ensure proper nerve alignment.

　　Transient Electrical Stimulation (BES): Following facial nerve injury, animals in groups 2 and 4 received brief electrical stimulation. The stimulation operation is adapted from Gordon et al.'s use of sciatic nerve stimulation in a rat model. Two wires coated with Teflon are exposed 2-3 mm outside. After the nerve is repaired, the first wire wraps around the proximal stump of the facial nerve. The second wire is embedded in the muscle tissue adjacent to the nerve and is located at the proximal end of the first wire. The insulated wire is connected to an isostim stimulator, which delivers 1.5 mA of current in 100 microsecond pulses at 20 Hz for one hour. The animal's right ear flapping confirmed the adequacy of the stimulation. After stimulation, the wire was removed from the animal and the wound was sutured.

　　Head implant surgery: After facial nerve surgery, complete head implant surgery under anesthesia. Make a small incision along the skull with a 15-blade scalpel from front to back. Blunt dissection fully reveals the lower skull. Using an electric drill, 4 holes were drilled in each quadrant of the skull, about 15 mm apart. Then place 1.6 mm screws at each drilling position. The dry acrylic resin is then liquefied and placed on the skull, covering the placed screws. Then pour two larger 5 mm threaded screws onto the threads before the resin solidifies.

　　Head and body fixation: Two weeks before the operation, all animal subjects received daily training. After the operation, the bodies of all experimental animals were fixed for one week. On the 14th day after surgery, the tentacles were measured. The test animal was initially given a small dose of isoflurane and transported to the body restraint device. They used screws through the exposed threaded screws for head fixation. The tentacles are marked and then placed on the mouse's face on either side. Once completed, introduce a scented stimulus for 5 minutes. Take the non-surgical left side as the control. Each rat completed this step at 2, 4, and 6 weeks after surgery.

　　Retrograde labeling of neurons: 3 months after surgery, carefully dissect and identify the buccal and marginal mandibular branches of the facial nerve again. The timeline of 3 months after surgery was selected as complete axonal regeneration, and regeneration is estimated to occur 10 weeks after injury. The cheek and marginal mandibular branches were severely severed, with a bifurcation of 5 mm. Each branch uses retrograde nerve tracer to identify each part innervated by motor neurons. Each nerve tracer is first placed in a small piece of gelatin sponge. The gelatin sponge was in contact with the nerve stump for 1 hour. Each nerve branch was washed extensively with saline. Take care to prevent cross marks. Each nerve tracer in the brainstem allows it to reach motor neurons for up to 4 days.

　　Cardiac perfusion tissue fixation: Animals are first injected with ketamine intraperitoneally. After incision of the abdominal cavity, the chest, ventricle and ascending and descending aorta are exposed. Using a No. 18 catheter, the left ventricle is penetrated and the catheter is inserted until the tip of the ascending aorta is seen. Then 300ml of 1M PBS was perfused through the catheter. Then infuse 400 ml PBS, and finally inject 4% paraformaldehyde through the catheter. After the animal was euthanized, the brain was exposed and removed. The brain samples were placed in 4% paraformaldehyde and then switched to 30% sucrose for 24 hours. After the tissue is cooled at -70 degrees, it is stored in a -80 degrees refrigerator.

　　Motor neuron count: The frozen tissue specimen has been taken out of the storage room, the frozen section is 20 microns thick, and the section is fixed on a glass slide and dried. After sectioning, observe with a fluorescence microscope. All motor neuron markers are only FR (red), only FG (blue), or both. The dividing cells were corrected by Abercrombie method and counted.

　　Result: All animals tolerated the operation without perioperative complications. They exhibited normal cage behavior and did not lose weight.

　　Measurement of functional results: All experimental animals experienced complete loss of ipsilateral vibration amplitude after surgery. In the second week, the average amplitude of group 1 (squeeze, no stimulation) was 14.4 degrees. Compared with the first group, the second group had an average amplitude of 24 degrees 2 weeks after surgery. The average swing amplitudes of groups 3 (transverse, no stimulation) and 4 (transverse, BES) were 4.8 and 14.6 degrees, respectively, which were statistically significant. In the fourth week, the first group showed the least amplitude loss, with an average of 11.6 degrees, while the second group had relatively unchanged amplitude from the second week, with an average of 23.2 degrees. The average amplitudes of the third and fourth groups in the fourth week were 9.1 degrees and 13 degrees, respectively. The average amplitude in group 1 was 20.3 degrees in the 6th week after surgery. The average amplitude of the second group was 26.7 degrees. There was no significant difference between groups 1 and 2 within 6 weeks after facial nerve surgery. Groups 3 and 4 showed similar amplitude at the 6th week. After 4 weeks of injury, BES significantly improved the swing ability of crushed animals. Two weeks after transection of the nerve, BES significantly improved the ability to swing.

　　Retrograde labeling of neurons: In control animals, FR (buccal branch) marked an average of 1388 neurons, while FG (mandibular margin) marked an average of 310 neurons. No double-labeled animals were observed in the control animals. Groups 1 and 2 had an average of 989 (49%) and 934 (46%) double-labeled motor neurons, respectively. Groups 3 and 4 had an average of 1299 (68%) and 1222 (62%) double-labeled neurons, respectively. Animals received BES (groups 2 and 4) to reduce the average double-labeled neurons after facial nerve injury. In general, compared with group 3 and group 4, BES significantly reduced group 1 and group 2 double-labeled neurons.

　　Conclusion: Studies have shown that transient electrical stimulation of the facial nerve crush injury model in rats is related to the acceleration of facial nerve function. BES can also induce specific regeneration of motor neurons after facial nerve injury. This has important clinical significance and potential application value for human facial nerve injury.