摘要
内耳微循环障碍是引起多种内耳疾病的主要致病因素之一。以往研究表明，突发性感音神经性聋、噪声性聋、老年性聋等都与内耳血流障碍有关。研究内耳微循环障碍对内耳功能和组织形态学的影响具有重要的临床意义。本研究通过建立豚鼠椎基底动脉缺血再灌注耳蜗损伤模型，观察了再灌注后各时间段耳蜗听功能及形态学的影响；运用TUNEL法、免疫组化法检测缺血耳蜗细胞凋亡情况及对凋亡相关蛋白bcl-2、bax进行分析，同时应用中药葛根素进行干预，探讨凋亡发生的可能机制及葛根素对缺血再灌注凋亡损伤的影响。研究发现，缺血再灌注造成豚鼠耳蜗明显听功能及形态学改变，TUNEL染色证实椎基底动脉缺血再灌注耳蜗细胞凋亡性损伤；葛根素对缺血再灌注耳蜗听功能及形态学有明显保护作用，同时能上调凋亡抑制因子bcl-2，下调凋亡诱导因子bax，从而达到抑制耳蜗缺血再灌注凋亡损伤的作用。
Objective The goal of this study was to establish an animal model of ischemia reperfusion injury of cochlea in guinea pigs and to observe the changes of morphology and auditory function,investigate the cell apoptosis and expression of bcl-2 and bax in the cochlear of guinea pigs after ischemia reperfusion in different time, analyze the effects of puerarin on the morphology,auditory function and cell apoptosis changes.
Methods Forty-eight healthy guinea pigs with normal Preyer ' s reflex weighed 200 to 250g were used in this study .They were randomly divided into eight groups: the normal control group, the sham operated group, the ischemia reperfusion groups (6, 24 hours, 7 days), puerarin groups(6, 24hours, 7days). The model of ischemia reperfusion injury in cochlea was established via the skull base approach. Under a surgical microscope,the clivus was gently drilled with a diamand bar. The dura was exposed and cut ,the arachnoid membrane covering the basilar artery was removed. After the basilar artery was exposed, the artery was occluded for 1 hour with a microvascular clamp,then the clamp was removed and the blood flow recirculated at the time point of 6 hours,24 hours and 7 days,respectively. The sham operated group was only exposed artery not occluded. In puerarin groups , puerarin 150mg/kg was given once a day by intraperitoneal injection. The auditory function was evaluated by evoked auditory brainstem responses(ABR).In each animal ,ABR was measured before the operation and after ischemia reperfusion, respectively. The histopathology changes of cochlea were observed by means of the Hematoxylin and Eosin(HE) staining under microscope . The cell apoptosis in the cochlea after ischemia reperfusion was investigated with terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling(TUNEL).The effect of puerarin on expression of bcl-2 and bax were studied by paraflin embedded immunohistochemistry.
Results
(1) The changes of ABR in the sham operated group were not evident; After ischemia reperfusion, all the wave latencies and interpeak latencies I -III of ABR were evidently prolonged, the auditory threshold was also increased, especially 24 hours after reperfusion. 7days after reperfusion, the auditory threshold partly resumed, but did not resume to the normal level .Puerarin could prevent effectively all wave latencies and interpeak latencies I -III of ABR from delaying by ischemia reperfusion injury and decreasing the auditory . threshold. A statistically significant difference was seen between the normal control group and the ischemia reperfusion groups, also between the ischemia reperfusion groups and puerarin groups(P<0.01).
(2) The changes of histopathology: The hair cells of the Corti,spiral ganglion cells(SGCs) and stria vascularis cells (SVC) were not found evident changes in the normal control group and the sham operated group. After ischemia reperfusion,outer hair cells were swollen,degeneration and defection, stria vascularis became thin, the number of SGCs decreased.24 hours after ischemia reperfusion, the changes became the most apparent. Compare with the ischemia reperfusion group, in puerarin groups,the degeneration and defection of outer hair cells were weaken, stria vascularis became a little thick, the number of SGCs increased.
(3) The negative immunohistochemical reaction of TUNEL staining was found in the hair cells,SGCs and SVC in the normal control group and the sham operated group. After ischemia reperfusion 6 hours, positive cells were found,with the rising time of ischemia reperfusion,there were significantly more positive cells,the peak time of positive cells expression was at 24 hours after reperfusion. In puerarin groups, positive cells decrease. The image analysis showed that the number of the TUNEL positive cells in the ischemia reperfusion groups was significantly increased than the normal control group's(P < 0.01).Between the ischemia reperfusion groups and puerarin groups, the TUNEL positive cells expression also had a statistically significant difference(P<0.01).
(4) The staining of bcl-2 and bax shows a lot of buffy yellow particles distributing in the cytoplasm and/or nucleus of the cochlear cells. The positive staining could be detected differently in the ischemia reperfusion groups and puerarin groups. The image analysis showed the downregulation expression of bcl-2 in the ischemia reperfusion groups,which was suppressed by puerarin. However, the upregulation of bax was showed in the puerarin groups,,and suppressed by puerarin.
Conclusions
(1) Cochlea ischemia reperfusion in guinea pigs could cause the lesion of cochlea,which was displayed auditory function and histopathology changes.
(2)TUNEL staining proved that the ischemia reperfusion injury manifested cochlear cells apoptosis damage,which was in accordance with hearing leision.
(3) Both bcl-2 and bax took part in adjusting cochlear cells apoptosis caused by ischemia reperfusion.
(4) Puerarin could relieve cochlear cells apoptosis damage caused by ischemia reperfusion.
引文
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