The Localization Of AQP1in The Facial Canal And The Relationship With Nerve Edema Of Facial Palsy Induced By HSV-1in Mice

【Background】 The original etiology of idiopathic facial nerve palsy is not clear, butglial edema and nerve entrapment are regarded as the main causes, and how long theedema lasts may directly determine the prognosis. And anatomical studies and clinical findingshas supported this hypothesis. Facial nerve trajecting in the facial canal is most vulnerable tooppress and swell.Although many research based on morphology or mechanics haveconfirmed the edema and high pressure in the facial nerve canal, the pathogenesis is lack offurther study, which is now construed as inflammation and ischemia-reperfusion. Asclinical treatment, facial nerve canal decompression and hormonotherapy are primarilyused in early stage of facial palsy. Though, the therapeutic effects of them are limited, theside effects are not easy to accepted. It is reported, more than40%of patients who sufferfrom facial palsy longer than three weeks will have permanent sequelae. As a result, amore simple and effective treatment for nerve edema is needed to be studied and resolvedurgently. Aquaporins(AQPs) are a family of membrane protein, which help maintainnormal water metabolism and transport. Once the AQPs get abnormal distribution orfunction, some diseases may occur. These years, the distribution and function of AQPssubtypes in the central nerve system(CNS) are well studied. The structure and function ofperipheral nervous system(PNS) are quite distinct from the CNS, as well as the AQPs. Inthe facial nerve, our Research Group has discovered the expression of AQP1. But thecellular localization and function are needed further investigation. We would like to findthe impact of AQP1in the facial nerve palsy, and regulate the edema to relieve theincarceration of facial nerve and the prognosis of these patients in the future.【Objective】(1) According to the microdissection and histological sections, locate thelabyrinthine segment of intratemporal facial nerve; afford a basis for study of theedematous ganglion geniculatum (GG);(2) Using the methods of immunofluorescence andWestern Blot, investigate the tissue and cellular distribution of AQP1in the full length ofintratemporal facial nerve；(3) Establish a mouse facial nerve palsy model induced byherpes simplex virus type1(HSV-1) inoculation; evaluate the features of the model bybehavioral assessment and histological facial motoneurons (FMNs) count; prepare a stableand controllable animal model for subsequent experiment;(4) By the methods ofimmunofluorescence, RT-PCR and Western Blot, detect the expression level of AQP1inthe facial canal at different time points, seeking the relationship between the nerve fiber edema and AQP1expresion.(5) Establish a cell cultrue colony of the intratemporal facialnerve, to verify the cellular localization of AQP1, afford a systemic basis for correlativeresearch of AQP1and Schwann cells.【Methods】(1) The cellular localization and full-length distribution of AQP1in the facial canal1. Divide15BALB/c mice(4-week-old, female,16~18g) into group A, B and Cequally. execute, perfuse and fix.2. In group A, mice were used for microdissection of the location, range and adjacentstructure of the intratemporal facial nerve. Afford an accurate location of gangliongeniculatum in the labyrinthine segment.3. In group B, mice were decalcified and sectioned continuously. Reserve twocontiguous sections in the position of the labyrinth segement of facial nerve to group twosets of sections. Sections I was stained by toluidine blue for microscopic observation thenerve edema of different segments.4. Sections II was stained by immunofluorescence of AQP1, S-100, IB-4and Hoechst,to ensure the tissue distribution and cellular localization of AQP1in the full length of theintratemporal facial nerve.5. In group C,5BALB/c mice were executed and the intratemporal facial nerve werecollected and cultrued. The immunofluorescence of AQP1, S-100and Hoechst was takento verify the expression of AQP1.(2) Estabilishment, improvment and assessment of the mouse facial nerve palsy modelinduced by HSV-11. Study the location, range and adjacent structure of extracranial facial nerve using5BALB/c mice, especially the posterior auricular branch, providing anatomical basis offacial palsy model induced by HSV-1.2. Divide30BALB/c mice(4-week-old, female,16~18g) into group I, group II and acontrol group in random, each group contain10Mice. And the facial nerve of left-side wasused for the experiment. In group I,25μl of virus solution (6.7×107plaque-forming unitsper milliliter; PFU/ml) was placed on the posterior surface of the left auricle after scratchedfor20times with a27-gauge needle. In group II, the titer of virus solution was9.5×106PFU/ml, and the virus solution was replaced by nutrient solution in the controlgroup.3. Detect the function of facial nerve by a behavior scale daily after the inoculation, compare the mortality, incidence and duration of facial palsy, find out the better modleestablishment method.4. At day28post-inoculation, mice of all groups were executed for FMNs countrespectively. The mice were perfused, fixed and the brains were sectioned continuously,then stained with toluidine blue in every fifth section and counted the number of the FMNs.Then, analyse the results of experimental and control sides in each group and betweengroups by using the student-t test, P <0.05, considering statistically significance.(3) The expression change and mechanism of AQP1in the intratemporal facial nerveafter facial palsy induced by HSV-1inoculation1. According to the different time points, divide52BALB/c mice(4-week-old, female,16~18g) into O1, O2(the inoculation day), A1, A2(9thday after the inoculation), B1, B2(16thday after the inoculation), C1, C2(40thday after the inoculation),5mice in O1andO2groups,7mice each in the rest group. Estabilsh the facial nerve palsy model throughthe method mentioned above. Mice from O1, A1, B1and C1groups were perfused, fixedand decalcified according to the time point of each group, then sentioned. Reserve twocontiguous sections in the position of the labyrinth segement of facial nerve to group twosets of sections: sections I and sections II.2. Sections I were stained by toluidine blue for calculating the ratio of facial nerve andfacial canal (FN/FC) to detect the edema level of facial nerve in the labyrinth segement.Then compare the tendency with expression of AQP1.3. Sections II were stained by immunofluorescence of AQP1and Hoechst to detect theexpression of AQP1in each time point.4. Mice from O2, A2, B2and C2groups were executed and the temporal segement offacial nerve tissue were fetched on the ice, then divided into two pieces after tissuehomogenate: one for Western Blot, and another for RT-PCR.5. Western Blot detection of the AQP1protein expression in the intratemporal facialnerve, and make statistics analysis to learn the trendency of the time-phase changes in thelevel of the AQP1protein expression in the intratemporal facial nerve.6. RT-PCR detection of the AQP1mRNA expression in the intratemporal facial nerve,and make statistics analysis to learn the trendency of the time-phase changes in the level ofthe AQP1mRNA expression in the intratemporal facial nerve.【Results】(1) Apply couples of double-label immunofluorescence technique, cell culture and identification. Discover the tissue and cellular localization of AQP1in the facial nerve forthe first time: AQP1was identified in the nerve fibers and vasa vasorum of intratemporalfacial nerve. No distributional difference of AQP1was observed in different segments ofthe facial nerve within the petrous bone. An obvious boundary was observed in the meatalsegment connecting the facial nerve nucleus with the peripheral portion. In the brain stempart of facial nerve, fibers were marked by AQP4, and there were no AQP1positive cells inthis segment. Distal to the boundary, there was only AQP1expressing. The boundary wasoverlapped with the CNS/PNS boundary, which also marks the alternation of two gial cells,the astroglia and Schwann cell. Cell culture identified the expression of AQP1in Schwanncell in vitro.(2) According to the regional anatomy study of the posterior auricular branch of facialnerve, successfully establish the acute and reversible facial palsy model by lowerconcentration and dose virus inoculation to the auricle surface after scratching. And therates of survival and success both were higher than before.(3) Through microdissection and slice observation, we ascertained the shape andlocalization of GG on the whole and slice; the edematous levels of facial nerve on differenttime points were evaluated by the ratio of FN/FC. It reached the culmination on the9thdaypost-inoculation, then reduced sharply, but were still higher than normal on16th day.(4) The expression of AQP1showed a kind of time-course change tendency in the progressof facial palsy induced by HSV-1kos strain inoculation. It reached the culmination at thesymptom appearence, then reduced sharply, but will not be normal untill3weeks after theinoculation.(5) Western Blot detection of the intratemporal facial nerve showed the expression ofAQP1protein reaching the peak value on the9thday post-inoculation, then reduced sharplytill the16thday, and gradually weakening until the40thday.(6) The results of RT-PCR showed the level of AQP1mRNA expression of theintratemporal facial nerve showed the same time-phase change in the Western Blotdetection..【Conclusions】(1) Confirm the availability of acute and reversible facial palsy model induced by HSV-1virus inoculation. Improve the modle by lower concentration and dose of virus inoculation,achieve lower mortality and higher rates of survival and success. This model can besuitable for the study of idiopathic facial palsy. (2) Compare the microdissection and Slice display of intratemporal facial nerve, ascertainthe shape and localization of ganglion geniculatum on the whole and slice. Afford asystemic basis for correlative research.(3) Confirm the tissue distribution and cellular localization of AQP1in the facial nerve forthe first time. And we also found its expression change in the level of protein and mRNAhighly coincide with the degree of the nerve edema in the facial canal, which hinted thenerve edema probably exist some intimate relationship with AQP1.(4) Verify the expression of AQP1in Schwann cell in vitro cell culture,and prepare forfruther mechanisms investigation.