Reconstruction Of Diaphragm Function Utilizing Nerve Transfer Technique In Rats Of Upper Cervical Spinal Cord Injury And Neural Circuits Plasticity

[Object] Phrenic motoneurons (PhMN) mainly locate in cervical 3-5 levels of spine cord. In upper cervical spinal cord injury patients, if damage involving the cervical 3-5 levels, then, on the other hand, a complete or incomplete diaphragm paralysis would occur. The impaired respiratory related muscle seriously limits the patient’s mobility, quality of life and phonation. Such patients in chronic progressing stage should adopt mechanical ventilation to assist breathing. However the complications of mechanical ventilation have gradually increased such as ventilator associated pneumonia, tension pneumothorax, and mediastinal emphysema etc. Respiratory related complications caused by prolonged mechanical ventilation are the main reason of the high mortality in patients with spinal cord injuries. Our research group found that anastomosis of the donor nerve originating above the level of spinal injury-spinal accessory nerve to ipsilateral phrenic nerve seems to be a promising surgery procedure reconstructing diaphragmatic function after cervical spinal cord injury. This research project proposes to establish animal model of unilateral third cervical spinal cord hemisection and to further explore diaphragm function recovery following nerve transfer technique. And neural circuits plasticity was studied utilizing neural retrograde tracer method.[Methods] 48 healthy adult male Sprague Dawley rats (100-150g), were randomly assigned into A, B, C three groups. Group A:blank control group; group B:left phrenic nerve and accessory nerve cut off at the clavian level; Group C:anastomosis of the donor nerve originating above the level of spinal injury-spinal accessory nerve to ipsilateral phrenic nerve at the clavian level. After 6 months,8 rats were randomly selected from each group, and received chest radiography, pulmonary function, and arterial blood gas analysis again. The other 8 rats in each group were then undertaken third spinal cord left hemisection injury. After 1 week, the 8 left rats were received chest radiography, pulmonary function, and arterial blood gas analysis. Another 24 healthy adult male Sprague Dawley rats (100-150g), were randomly assigned into D, E, F three groups. Group D and E:healthy rats group; group F:anastomosis of the donor nerve originating above the level of spinal injury-spinal accessory nerve to ipsilateral phrenic nerve at the clavian level. After 6 months, rats in group E and F were undertaken third spinal cord left hemisection. Green fluorescent protein (GFP) marked gene recombinant pseudo rabies virus was then injected into 4 sits of left hemi diaphragm clock wisely, each point 10μl.4 days later, the rats were perfused transcardiacally, and the brains were removed from skulls. Then it was cut into serial frozen sections with cryostat, and observed by fluorescence microscope. The sections with fluorescence were selected, and compared with "The rat brain in stereotaxic coordinates" then stained by immunohistochemistry. The images were acquired using NIS-Elements 2.1 software, and staining nucleus was counted by Image-Pro Plus 6.0 software. Statistical analysis was made by SPSS 20.0 software, using the single factor analysis of variance (one-way ANOVA) between groups. Bonferroni method (constant variances) or the Mann-Whitney method (unequal variances) was used in pairwise comparison.[Results] Before third level cervical spine cord left part hemisection, pulmonary function index in group B and C are both lower than in group A. group C is better than that of group B. After the hemisection, pulmonary function index in group A decreased more than 50%, slightly lower than that of group B. While the indexes were basically flat in group B and group C. No fluorescence imaging of neurons were detected in C3 spinal cord hemisection rats of group E, while in group D and F, fluorescence was detected in the Medulla. Immumohistochemical staining testified that the fluorescence was from green fluorescent protein marked gene recombinant pseudo rabies virus.[Conclusions] Nerve transfer method-anastomosis of the donor nerve originating above the level of spinal injury-spinal accessory nerve to ipsilateral phrenic nerve is a promising surgery procedure reconstructing diaphragmatic function after upper(C3-C5) cervical spinal cord injury. It can partially improve the diaphragmatic function, restoring diaphragmatic muscle contraction. Through nerve transfer, diaphragm build up new connection with medulla oblongata, the neural circuits played an important role in functional recovery of the rat diaphragm.