阿米卡星對(duì)豚鼠耳蝸毛細(xì)胞的損傷作用

1、阿米卡星對(duì)豚鼠耳蝸毛細(xì)胞的損傷作用                           作者：劉雙月,王愛(ài)梅,許濤,牟華【摘要】  目的 研究阿米卡星(amikacin，AMK)在不同給藥時(shí)間內(nèi)對(duì)豚鼠耳蝸毛細(xì)胞的損傷作用，探討AMK的耳毒性。方法 豚鼠隨機(jī)分為對(duì)照組、AMK 3 d組、AMK 7 d組和AMK 11 d組。采用異

2、硫氰酸四甲基羅丹明標(biāo)記的鬼筆環(huán)肽熒光染色方法，并結(jié)合聽(tīng)腦干反應(yīng)(auditory brainstem response，ABR)測(cè)試，觀察用藥前后耳蝸毛細(xì)胞形態(tài)及聽(tīng)功能的改變。結(jié)果 AMK首先損傷耳蝸底回的外毛細(xì)胞,并且隨著給藥時(shí)間延長(zhǎng)，損傷逐漸向頂回發(fā)展，外毛細(xì)胞缺失明顯增多，而AMK對(duì)內(nèi)毛細(xì)胞的損傷要輕于外毛細(xì)胞。聽(tīng)功能改變與形態(tài)學(xué)變化基本一致。結(jié)論 AMK可損傷豚鼠耳蝸毛細(xì)胞，導(dǎo)致聽(tīng)功能下降。 【關(guān)鍵詞】  阿米卡星;豚鼠;耳蝸;聽(tīng)腦干反應(yīng)           

3、0; Impairment Effect of Amikacin on Cochlear Hair Cells in Guinea PigLIU Shuangyue, WANG Aimei, XU Tao, MU Hua(Department of Physiology, Liaoning Medical University, Jinzhou 121001 China)Abstract:Objective To investigate the impairment effect of amikacin (AMK) on cochlear hair cells in guinea pig at

4、 different injection time, and to explore ototoxicity of AMK. Methods Guinea pigs were randomly assigned to four groups: control group, AMK 3-day group, 7-day group and 11-day group. Tetramethylrhodamine isothiocyanate-labeled phalloidine staining and auditory brainstem response (ABR) measurement we

5、re used to evaluate the changes of hair cells morphology and auditory function before and after the drug treatment. Results Outer hair cells (OHCs) in the basal turns were first impaired by AMK, and the impairment of OHCs developed to the apical turns with time prolonging, and the loss of OHCs was i

6、ncreased significantly. The injury of inner hair cells (IHCs) induced by AMK was lighter than OHCs. Change of auditory function was basically consistent with that of morphology. Conclusions AMK can damage the cochlear hair cells, leading to decline in auditory function.Key words:amikacin; guinea pig

7、; cochlea; auditory brainstem response氨基糖苷類抗生素(aminoglycoside antibiotics，AmAn)具有強(qiáng)大的抗革蘭氏陰性桿菌的作用, 加之價(jià)格低廉, 是臨床控制感染的常用抗生素1。但是AmAn具有嚴(yán)重的耳毒性，可導(dǎo)致耳蝸毛細(xì)胞、前庭毛細(xì)胞及螺旋神經(jīng)節(jié)細(xì)胞的損傷2-3。阿米卡星(amikacin, AMK)為半合成的AmAn，亦可對(duì)耳蝸毛細(xì)胞造成不可逆性的損傷4，然而AMK在不同給藥時(shí)間內(nèi)對(duì)豚鼠耳蝸毛細(xì)胞的損傷特點(diǎn)及其與聽(tīng)功能變化的關(guān)系，目前尚不清楚。據(jù)此，本研究應(yīng)用異硫氰酸四甲基羅丹明(Tetramethylrhodamine iso

8、thiocyanate，TRITC)標(biāo)記的鬼筆環(huán)肽熒光染色方法，并結(jié)合聽(tīng)腦干反應(yīng)(auditory brainstem response，ABR)測(cè)試，觀察用藥前后豚鼠耳蝸毛細(xì)胞形態(tài)及聽(tīng)功能的改變，以探討AMK的耳毒性。1 材料與方法1.1 實(shí)驗(yàn)動(dòng)物健康白毛紅目豚鼠47 只，體重250300 g，雌雄不限，耳廓反射靈敏，無(wú)中耳炎，由遼寧醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供。實(shí)驗(yàn)期間所有豚鼠均在安靜狀態(tài)下飼養(yǎng)并給予常規(guī)飲食。1.2 主要試劑和儀器阿米卡星注射液(0.1 g/mL，批號(hào)090327)由蘇州第六制藥廠生產(chǎn)，TRITC-鬼筆環(huán)肽購(gòu)自美國(guó)Sigma公司，Smart EP & OAE聽(tīng)覺(jué)誘發(fā)電位

9、-耳聲發(fā)射記錄系統(tǒng)由美國(guó)智聽(tīng)公司生產(chǎn)，ZEISS A1熒光顯微鏡由德國(guó)Zeiss公司生產(chǎn)。1.3 動(dòng)物分組與耳毒模型建立將動(dòng)物隨機(jī)分成4 組，即對(duì)照組10 只，AMK 3 d組11 只，AMK 7 d組12 只，AMK 11 d組13 只。以上4 組中，AMK 3 d組、7 d組和11 d組每天肌肉注射AMK 400 mg/kg，分別連續(xù)給藥3 d、7 d和11 d;對(duì)照組則每日肌肉注射等量生理鹽水。用藥期間監(jiān)測(cè)動(dòng)物體重以調(diào)整藥量。1.4 ABR測(cè)試各組動(dòng)物于用藥前及停藥后24小時(shí)內(nèi)分別測(cè)試ABR。操作在隔音屏蔽室內(nèi)進(jìn)行。豚鼠用1 %戊巴比妥鈉40 mg/ kg 經(jīng)腹腔麻醉后，將電極的正極置于

10、動(dòng)物顱頂正中皮下，負(fù)極置于給聲側(cè)耳廓后下，接地電極置于對(duì)側(cè)耳廓后下，屏蔽耳機(jī)距測(cè)試豚鼠外耳道口0.5 cm。應(yīng)用Smart EP & OAE聽(tīng)覺(jué)誘發(fā)電位-耳聲發(fā)射記錄系統(tǒng)給予短純音(tone burst)刺激, 選取4、12、24 kHz 3個(gè)頻率分別進(jìn)行測(cè)試并記錄ABR閾值(聽(tīng)閾)。重復(fù)率39.10次/s，帶通濾波1001 500 Hz，疊加1 024 次，掃描時(shí)程16.0 ms。聲刺激強(qiáng)度從95 dB SPL開(kāi)始，以5 dB逐次遞減，聽(tīng)閾判定以剛出現(xiàn)ABR 波為準(zhǔn)并至少重復(fù)兩次。同一刺激頻率下給藥前后的聽(tīng)閾差值記為ABR閾移。1.5 耳蝸鋪片TRITC-鬼筆環(huán)肽熒光染色各組豚鼠于最

11、后一次ABR測(cè)試結(jié)束后，快速斷頭，取出聽(tīng)泡。解剖顯微鏡下去除鐙骨，打開(kāi)前庭窗和蝸窗，用含有4%多聚甲醛的0.1 M PBS(pH 7.4)灌流3 次，并置于該溶液中4 下固定24 h。然后將標(biāo)本置于8 % EDTA中脫鈣23 d。PBS漂洗2 次后，于解剖顯微鏡下分離全耳蝸基底膜。將基底膜置于0.25% Triton X-100溶液中浸泡50 min，然后進(jìn)行TRITC-鬼筆環(huán)肽染色45 min。PBS漂洗后分回鋪片，以熒光封固劑封片。熒光顯微鏡下觀察各回毛細(xì)胞損傷情況。1.6 統(tǒng)計(jì)學(xué)分析應(yīng)用SPSS 16.0統(tǒng)計(jì)軟件包對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析，數(shù)據(jù)以±s表示。各組之間ABR閾移的比較采用單因素方差分析，P&