海人酸致鸡耳蜗传入神经元迟发性死亡

Delayed neuron death of chicken' s cochlear afferent neurons induced by kainic acid

目的 观察不同剂量谷氨酸(Glu)类似物海人酸(kainic acid,KA)导致鸡基底乳头(以下称为叫“耳蜗”)传入神经元迟发性神经元死亡(delayed neuron death,DND)及相应的耳蜗电生理改变,探讨兴奋性神经元死亡的发生规律。方法 34只成年来亨鸡分3组。第一组经左耳蜗鼓阶灌注高剂量KA(5 mM,KA-H组)后测量不同时点的CAP、CM和DPOAE,并观察耳蜗显微和超微结构变化;第二组灌注低剂量KA(0.3mM,KA-L组)后只观察各时点耳蜗形态变化;第三组灌注Hank氏液(HBSS)作对照。结果 ①KA灌注后,KA-H和KA-L组高毛细胞(THC)下方的传入树突均迅速水肿,CAP振幅大幅下降;矮毛细胞(SHC)的形态不受影响。②KA灌注后,KA-H组大量传入神经元于2～4周后发生DND并有不可逆听力损伤,但THC长期存活;面KA-L组传入神经元数目无明显减少,且神经元和髓鞘病变在1周后开始恢复。结论 ①KA以剂量依赖性方式选择性损伤鸡耳蜗传入神经元;局部高浓度可造成DND,其发生的时间在KA处理2～4周后;②鸡耳蜗THC可在没有传入突触联系的情况下长期存活,这可能用作实验模型;③为推测人类耳蜗兴奋性损伤及其机理,有必要用生理性递质Glu对哺乳动物进行在体实验。

Objective To observe the delayed neuron death (DND) in excitotoxically damaged cochlea of chickens caused by kainic acid (KA), a potent analog of glutamate, with different dosages. Methods Thirty-four adult white Leghorn chickens were divided into 3 groups. Group 1: Perilymphatic perfusion of KA (5mM, 10μl, KA-H) was followed by electrophysiological determinations of compound action potential (CAP), cochlear microphonic (CM) and distortion product otoacoustic emissions (DPOAE) at different time points from 30 minutes to 20 weeks. Simultaneous light and electron microscopy were then taken at the same time points. Group 2; Perilymphatic perfusion of KA (0.3 mM, 10 uuuul, KA-L) was followed by light and transmission electron microscopy at different time points from 30 minutes to 4 weeks. Group 3; Perilymphatic perfusion of Hank's balanced salt solution (HBSS) was perfused as the control. A newly developed round window implanted electrode was used to record CAP and CM. The CAP, CM, and DPOAE were recorded before and after the perilymphatic perfusion in the KA-H group. Results The swelling of the afferent dendrites beneath the tall hair cells (THC) rapidly occurred within 30 minutes after the KA treatment in both the KA-H and KA-L groups without any morphological changes in short hair cells (SHC) and efferent endings. Meanwhile, a rapid and large reduction of CAP amplitude was observed less than 30 minutes after the KA treatment in the KA-H group, but CM and DPOAE remained unchanged. Afterwards, the mass of DND and irreversible hearing loss were observed in KA-H group, whereas the THC remained almost intact even at 20 weeks post KA perfusion. Nevertheless, no sign of DND was found in the KA-L group. Conclusion The results suggest that ① KA selectively damages the afferent neurons of chicken's cochlea in a dose dependent manner. DND may result from high regional concentration of excitotory amino acid, such as KA. ② KA induced DND, in our experiment system, may occur around 4 weeks post-KA treatment. ③ Chicken's THC can be kept alive without afferent innervations. ④ in vivo experiment with glutamate in mammals is reasonably needed to explore if similar DND would occur in the human cochlea and its mechanisms under normal and/or pathological conditions. Several experimental findings indicated that might occur in irreversible sensorineurial hearing loss caused by anoxia or acoustic cochlear injury. The objective of this paper was to observe the DND in excitotoxic damaged cochleae of chickens caused by kainic acid (KA), a potent analog of glutamate, under different dosages.