The Changes In The Degree Of Nerve Edema And The Level Of AQP1 Expression In The Facial Canal After Facial Nerve Axotomy

【Background】Facial nerve exits from the stylomastoid foramen of petrous pyramid and then courses superficial. The facial nerve is easily exposed to facial trauma or to damage during operations in adjacent regions, resulting in disordered motor function that affects the muscles of facial expression and negatively impacts the patient's quality of life. Therefore, the repair of facial nerve injury has been studied intensively, and the main direction of those studies are always on the coup injury in the facial nerve nucleus and axons by various physical, chemical and biological stimuli. However, there are few studies focused on the formation merchanism of secondary edema of facial nerve causing by various wound agents and the posibility of the injury induced by subsequent vicious cycle of'edema- ischemic-edema'similar with the Bell's palsy in the facial canal which is tortuous and narrow,as well as rigid. Aquaporins (AQPs) are a family of small integral plasma membrane proteins that primarily transport water across the plasma membrane. There are 13 members (AQP0-12) in humans and distribute in several tissues and organs of entired body. It is confirmed that AQPs have affinity with the formation and elimination of edema in the related tissues and organs. In the peripheral nerveous system (PNS), it is reported that AQP1 is the one mainly expressed and distributed. However, its function and mechanism in PNS is not yet clear. In our study, we intend to preliminarily comfirm the expression and the distribution of AQP1 in the facial canal, and observe the change in the degree of nerve edema and the level of AQP1 expression at different time points after facial nerve axotomy. According the analysis to the results, we attempt to preliminarily investigate the relationship of AQP1 and nerve edema, which will provide theoretical basis for the subsequent research on the effect and mechanism in the edema injury of facial nerve.【Objective】(1) Establish a mouse facial nerve axotomy model involving the subauricular incision approach; evaluate the effect and the outcome of the facial nerve transection by electroneurophysiology, behavioral assessment, and histological observation; afford a stable animal model for the subsequent experiment; (2) According to the observation of histological sections, compare the change in the degree of nerve edema in the facial canal at different time points; (3) By the methods of immunohistochemistry, Western Blot and RT-PCR, compare the change in the level of AQP1 expression in the facial canal at different time points; (4) explore the possible roles of AQP1 in the facial canal towards to the nerve edema.【Methods】(1) Estabilishment and assessment of the mouse facial nerve axotomy model1. Study on the regional anatomy of facial nerve and adjacent tissue in BALB/c mice to determinate the incision approach and surgical technique.2. Divide 20 mice into an operation group and a sham operation group in random, and in both groups, the left-side facial nerve was used for the operation, and the right side served as the control; establish the facial nerve axotomy model through the subauricular incision approach.3. Detect the compound muscle action potentials (CMAP) of the operated-side vibrissa pad-muscles in all of experimental mouse before and after transection of the facial nerve and on the 21st day post-operatively, and evaluate the facial nerve function preoperatively and at the 1st, 3rd, 7th, 14th and 21st days postoperatively.4. At day 28 postoperatively, perfuse the mice, fix the brain,and take continuous frozen sections of brainstem containing facial nucleus, then stain with toluidine blue in every fifth section and count the number of the facial motoneurons (FMNs), at last, analyse the results of both operation and control sides in each group by using the student-t test, P <0.05, considering statistically significance.(2) The variation trendency of the degree of nerve edema in the facial canal1. study on the regional anatomy of the segements of facial nerve in the facial canal of mouse, and take continuous frozen sections of the temporal bone containing the facial canal after decalcification to observe the morphology of the segements of facial nerve in the facial canal in section; according to the above results, determinate the observed level in our experiment.2. According to the different postoperative time points, divide 30 BALB/c mice into A(immediate postoperation), B(postoperatively 6 hours), C(postoperatively 1 day), D(postoperatively 3 days), E(postoperatively 5 days), and F(postoperatively 7 days) groups, 5 in each group. Estabilsh the the facial nerve axotomy model through the subauricular incision approach, then perfuse, fix and decalcify according to the time point of each group.3. Take continuous frozen sections of the decalcified temporal bone containing the facial canal, and reserve two contiguous sections in the position of the labyrinth segement of facial nerve in the facial canal to group two sets of sections: one in observing the nerve edema and another in observing the expression of AQP1 in the next stage of this experiment.4. Calculate the ratio of the cross-sectional area in the facial nerve and the facial canal (FC/FN), and make statistics analysis to learn the trendency of the time-phase changes in the degree of nerve edema in the facial canal.(3) the trendency of the changes in the level of AQP1 expression in the facial canal1. Immunohistochemical staining for AQP1 in the set of section from the above experimental stage, observe the distribution and expression of AQP1, and preliminary analyse the time-phase changes of AQP1.2. Divide 30 BALB/c mice into six groups by the method described in above. Establish the axotomy model through the subauricular incision approach, and according to the time point of each group, achieve the temporal segement of facial nerve tissue on the ice, then divide two pieces after tissue homogenate: one for Western Blot, and another for RT-PCR.3. Wester blot detection of the AQP1 protein expression in the temporal segement of facial nerve, and make statistics analysis to learn the trendency of the time-phase changes in the level of the AQP1 protein expression in the temporal segement of facial nerve.4. RT-PCR detection of the AQP1 mRNA expression in the temporal segement of facial nerve, and make statistics analysis to learn the trendency of the time-phase changes in the level of the AQP1 mRNA expression in the temporal segement of facial nerve.【Results】(1) According to the study on the regional anatomy of the extra cranium segement of facial nerve, successfully establish the mouse facial nerve axotomy model involving the subauricular incision approach;(2) According to the observation of the serial section of the facial canal, confirm the location of the labyrinth segement of facial nerve in the facial canal; from the value of FN/FC of the labyrinth segement in different groups, learn the degree of nerve edema in the facial canal in different time points, and according to the results, we found the nerve edema in the facial canal was increasing from the 6th hour postoperatively and reach the peak value at the 3th day after surgery, then the degree of edema would gradually vanish until the 7th day postoperatively.(3) Detecting by the methods of immunohistochemistry staining, Western Blot, and RT-PCR, we first confirmed the distribution and expression of AQP1 in the temporal segement of the facial nerve; immunohistochemistry staining showed AQP1 crico-coiling around the nerve fiber as hollow pipe in the labyrinth segement, and presenting the time-phase change after axotomy.(4) Western Blot detection showed the expression of AQP1 protein enhancing in the 6th hour postoperatively, reaching the peak value at the 3th day, and gradually weakening until the 7th day.(5) The results of RT-PCR showed the same time-phase change in the level of AQP1mRNA expression.【Conclusions】(1) Compared with the classic postauricular incision approach, we found that the operation procedure adopted the subauricular incision approach not only could attained the stable experimental results, but also possesses multiple advantages including minimal invasion, easy exploration and full exposure of the facial nerve, as well as providing a larger operation space.(2) Observe the nerve edema in the facial canal and definite the position for observation; we found the injury of axotomy could cause the secondary edema in the proximal end of the facial nerve, and go through the process of gradual change from ingravescence to extinction, in which the time point for the most evidental edema is three days after injury. It provides the experimental data for the research in the edema injury mechanism.(3) It is the first time that confirm the distribution and expression of AQP1 in the facial nerve. And through this study, we also found its expression change in the level of protein and mRNA highly coincide with the dgree of the nerve edema in the facial canal, which hinted the nerve edema probably exist some intimate relationship with AQP1.