| [Background]Facial nerve palsy can be divided into central and peripheral one according to the injury site. An important reason for the high morbidity of peripheral facial paralysis which lacks efficient treatment is decided by the unique anatomy structure. Facial nerve which is derived from the brain and enters the internal auditory canal begins a journey through a long and narrow bony canal. The lesion once initiates in the canal, the inflammation of facial nerve starts the edema process. Due to the restriction of the bony canal, the facial nerve has little room to swell and in return increases the pressure of the canal. Raised pressure in the canal leads to severe ischemia and the’edema-ischemic-edema’cycle which leads to a severer nerve lesion. During the course of facial paralysis, edema plays an important role. And reducing the edema of facial nerve is a key point. Aquaporins are a kind of membrane channel proteins which can be used to help water molecules get through the membrane rapidly and it participated in water metabolism in multiple body tissue and organs. Our previous Studies also confirmed that AQP1is expressed in Schwann cells of mouse facial nerve. And in the transection injury model of mouse facial nerve, we find that AQPl expression was significantly increased after the nerve edema caused by the injury and it suggests that AQP1may be involved in the process of nerve edema. So far, there is no drug or compounds are confirmed to regulate the expression of AQP1. Acetazolamide as a possible AQP1inhibitor also have been reported, but there exists some controversy. So this study intends to explore the regulatory effects of acetazolamide on AQP1expressed in the facial nerve of mouse, and whether there is a long term or a short term effect is observed. In the facial nerve transection injury model of mouse, nerve edema and AQP1expression is observed after given of acetazolamide, in order to provide a suitable way for the treatment of facial nerve edema.[Objective]1. Try to find the regulation effect of acetazolamide on the expression of AQP1of Schwann cell at cellular level.2. The osmotic pressure experiment was performed to find the regulation of acetazolamide on the function of AQP1of Schwann cell at cellular level.3. In the mouse facial nerve axotomy model, Facial nerve edema and AQP1expression were detected to find the effect of AZA during the edema process after the injury. And try to provide a new idea for clinical treatment of facial nerve edema. [Methods]1. Try to prove the expression of AQP1on primary cultured Schwann cells of facial nerve.(1) Both sides of facial nerve in5-days-old new born Balb/c mice were cut off and digested by trypsin, and the cells were purified by a difference-speed adherence method.(2) AQP1expression in Schwann cells were analyzed by a double immune staining of AQP1and S-100.2. MTT detection was used to find out the toxic effects of acetazolamide on proliferationon of cultured Schwann cells of facial nerve.3. Water permeability (Pf) was measured through the osmotic pressure experiment to learn the effect of AZA lay on the Schwann cells in short term.The primary cultured Schwann cells were divided into3groups. Group A:the primary cultured Schwann cells with no drugs; Group B:a final concentration of10μM AZA were added for30min before the experiment; Group C:a final concentration of10μM HgCl2was added for30min before the experiment. After added calcein-AM, Schwann cells were incubated for30min in37℃dark environment, the fluorescent agent was permeated into the living cells. Then when we changed the liquid environment outside the cell membrane suddenly into low permeability, the H2O molecules went into the cells and the fluorescent agent was dilated. We used the confocal microscopy to record the changes of the intracellular fluorescence intensity, and obtain the water permeability. While the value of Pf directly reflects the effects of AZA on Schwann cells in short term.4. A final concentration of0.1μM、1μM、10μM of AZA were added in cultured Schwann cells, and we used the RT-PCR and Western Blot detection to observe the change of mRNA and protein expression of AQP1, trying to find the long term effects of the drugs on the expression of AQP1.5. The5weeks old healthy Balb/c mice weighed20-22g were selected to construct the facial nerve axotomy model. Nerve in the facial nerve canal were observed and also the degree of nerve edema and expression of AQP1.(1) We established the facial nerve axotomy model through the subauricular incision approach, and transect the nerve in front of the Cartilage. The experimental group were given AZA (40mg/Kg·d) intragastrically, and the control group0.9%normal saline (40mg/Kg·d). At the day1、day3、day5、day7after transection, the mice were killed to get the intracranial segment of facial nerve. RT-PCR and Western Blot detection was performed to analyze the effect of AZA on the mRNA and protein expression of AQP1.(2) After the facial nerve axotomy model was established, the experimental group and the control group were treated the same as above. At the day1、day3、day5、day7after nerve transection, the skulls were cut off and fixed by4%paraformaldehyde and decalcified by10%EDTA and dehydrated by20%-30%saccharose. Then we took the continuous frozen sections of the skull to learn the effect of AZA on the edema of facial nerve.[Results]1. A double immune staining of AQP1and S-100were performed on the primary cultured Schwann cell of Balb/c mouse, and both were observed through the fluorescence microscope. The AQP1expression on the Schwann cell is confirmed.2. No significant effects of acetazolamide on cultured Schwann cell proliferation were found.3. In the hypotonic perfusion experiment, the perfusion curve of the control group (group A) was a typical "Z" curve, showing a good permeability for water. The value of Pf is largest among the4groups. The perfusion curve was also a typical "Z" after the hypotonic shock in group B which Schwann cells incubated in10μM final concentration of acetazolamide for30min. And the value of Pf is smaller than that of group A. No "Z" curve was seen in group C which Schwann cells incubated in10μM final concentration of HgC12for30min. The values of Pf of group C were much smaller than that of group A and group B, and the results showed Group A>Group B>Group C.4. In the primary cultured Schwann cells with different concentration of acetazolamide co-cultured for72h, results of Western and RT-PCR test shows that in the final concentration of0.1μM, acetazolamide can significantly reduce the expression of AQP1in the level of gene and protein with no obvious sign of concentration dependent.5. In the facial nerve amputation model, mice acetazolamide was given acetazolamide (experimental group) and normal saline (control group) respectively. The expression of AQP1was detected by both Western Blot and RT-PCR. The results of Western Blot showed that AQP1expression in experimental group lower than that of the control group on day3, and higher than that of control group on day7. The results of RT-PCR showed that AQP1mRNA expression in experimental group lower than that of the control group on day1and day3, while higher than that of control group on day5、7.6、Anatomy of the facial nerve in the skull of mice:Facial nerve enters the facial nerve canal right after the nerve stemmed out from the brain, and the nerve takes its two sharp turns near the entrance and exit of skull. The shape of facial nerve in skull is like an "S". The aqueductus cochlease vein goes with the facial nerve in the cochlea section, and the facial nerve canal is not a continuousely closed bony canal. When the facial nerve enters the labyrinthine segment, the facial nerve canal turns back to be a continuousely closed bony canal.7. The facial nerve transaction model in mice. Observing and measursing the edema of intratemporal facial nerve by frozen section showed the results that the edma of facial nerve was obvious in day1after the nerve transaction and in day3the edema reached its peak. Nerve swelling showed a lesser degree of AZA treated mice after transection of the facial nerve in the experimental group than in the control group, but in day1, day5and day7nerve swelling between the two groups showed no significant difference.[Conclusions]1. Acetazolamide had no obvious effect of inhibition on the growth of Schwann cells.2. No obvious inhibitory effect on primary cultured Schwann cells were found by acetazolamide.3. Acetazolamide had negative short and long term effect on the regulation of primary culture Schwann cells.4. In the facial nerve amputation model, acetazolamide can suppress the peak expression of AQP1and also reduce facial nerve edema peak level (3rd day after injury), the effect is not obvious at the period of recovery. |
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