听觉脑干植入豚鼠模型构建的标准化步骤及评价

doi:10.3969/j.issn.1674-8115.2022.05.005

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (5): 583-590.doi: 10.3969/j.issn.1674-8115.2022.05.005

听觉脑干植入豚鼠模型构建的标准化步骤及评价

周祥¹^,²(), 潘金锡¹^,², 张钦杰¹^,², 李蕴¹^,²^,³, 陈颖¹^,²^,³, 谭皓月¹^,², 彭飞², 黄穗⁴, 谭治平⁴, 吴皓¹^,², 贾欢¹^,²()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海市耳鼻疾病转化医学重点实验室，上海 200125
^3.上海交通大学医学院附属第九人民医院听觉植入中心，上海 200125
^4.浙江诺尔康神经电子科技股份有限公司，杭州 311100

收稿日期:2022-03-27 接受日期:2022-04-15 出版日期:2022-05-28 发布日期:2022-05-28
通讯作者: 贾欢 E-mail:zhouxiang0618@sjtu.edu.cn;huan.jia.orl@shsmu.edu.cn
作者简介:周祥（1991—），女，住院医师，硕士；电子信箱：zhouxiang0618@sjtu.edu.cn。
基金资助:
国家自然科学基金青年科学基金(82000990);上海市黄浦区产业扶持基金(XK2019015);上海市人才发展基金(2019047);上海市耳鼻疾病转化医学重点实验室(14DZ2260300);上海交通大学医学院转化医学协同创新项目(TM202011)

Establishment and evaluation of standardized steps for building a guinea pig model of auditory brainstem implantation

ZHOU Xiang¹^,²(), PAN Jinxi¹^,², ZHANG Qinjie¹^,², LI Yun¹^,²^,³, CHEN Ying¹^,²^,³, TAN Haoyue¹^,², PENG Fei², HUANG Sui⁴, TAN Zhiping⁴, WU Hao¹^,², JIA Huan¹^,²()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200125, China
^3.Hearing Implantation Center, Shanghai Ninth People's Hospital, Shanghai 200125, China
^4.Nurotron Biotechnology Co. , Ltd. , Hangzhou 311100, China

Received:2022-03-27 Accepted:2022-04-15 Online:2022-05-28 Published:2022-05-28
Contact: JIA Huan E-mail:zhouxiang0618@sjtu.edu.cn;huan.jia.orl@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(82000990);Shanghai Huangpu District Industrial Support Fund(XK2019015);Shanghai Talent Development Fund(2019047);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300);Collaborative Innovation Project for Translational Medicine at Shanghai Jiao Tong University School of Medicine(TM202011)

摘要/Abstract

摘要：

目的·经听泡-迷路径路改良建立人工听觉脑干植入（auditory brainstem implantation，ABI）豚鼠模型并评价该模型的有效性和可靠性。方法·对20例听力正常的雄性健康成年白化豚鼠行改良后经听泡-迷路径路ABI手术，术中暴露脑干处耳蜗背侧核（dorsal cochlear nucleus，DCN）后植入豚鼠专用ABI电极阵列，记录电诱发听性脑干反应（evoke auditory brainstem response，eABR），并与术中耳蜗内eABR记录波形进行比较。留置ABI电极后，于术后48 h、7 d、30 d、180 d行安乐死前记录DCN处eABR波形；取出含电极脑组织，行苏木精-伊红染色、免疫荧光染色观察神经元、胶质、小胶质细胞的变化；定量PCR检测白介素-1β（interleukin-1β，IL-1β）和基质金属蛋白酶9（matrix metalloprotein-9，MMP-9）的表达。结果·术中所有动物均成功引出DCN处eABR，术后死亡时间集中在电极植入后36~48 h。DCN处eABR波形形态与耳蜗处eABR相似，DCN处eABR中Ⅲ波相比耳蜗处eABR中Ⅳ潜伏期缩短（0.84±0.22）ms（n=19），DCN处较耳蜗处平均阈值减小（0.11±0.14）mA（n=19）。术后DCN处eABR与术中比较，阈值平均增加（0.10±0.10）mA（n=16），Ⅲ波潜伏期平均延迟（0.15±0.21）ms（n=16）。组织学形态分析显示电极周围的耳蜗核接触面在术后48 h出现空泡样变性，术后48 h、7 d及180 d在DCN边缘均可见胶质成分增多。定量PCR结果显示IL-1β、MMP-9基因表达量在48 h与7 d时高于未植入电极豚鼠，但差异无统计学意义。结论·建立的ABI植入豚鼠模型经实验评估，建模成功率高且重复性好；初步观察到电极神经界面出现周围胶质及纤维成分增生。

关键词: 人工听觉脑干植入, 动物模型, 豚鼠, 听觉电生理, 组织学形态

Abstract:

Objective·To establish the guinea pig model of auditory brainstem implant (ABI) by modified transbulla-labyrinthine approach and assess its effectiveness and reliability.

Methods·Twenty healthy adult male albino guinea pigs with normal hearing underwent ABI surgery via modified transbulla-labyrinthine approach. During the operation, the dorsal cochlear nucleus (DCN) of the brainstem was exposed laterally and implanted with a special ABI electrode array for guinea pigs. The evoked auditory brainstem response (eABR) was recorded from DCN and compared with the eABR waveform elicited from cochlear intraoperativly. After the electrode was implanted, the eABR waveform at DCN was recorded before euthanasia at 48 h, 7 d, 30 d and 180 d after operation. Then the brain tissue containing the electrode was harvested and the electrode neural-interface was evaluated by hematoxylin-eosin staining, immunofluorescence staining and q-PCR.

Results·The eABR was successfully recorded at DCN from all animals during surgery, and the death occurred at 36 h to 48 h after operation. The waveform morphology of eABR at DCN was similar to that at cochlea. The latency of wave Ⅲ in eABR at DCN [ (0.84 ± 0.22)ms] was shorter than that of wave Ⅳ in EABR at cochlea (n=19), and the threshold difference between them was (0.11±0.14) mA (n=19). Compared with the intraoperative eABR at DCN, the average threshold increased by (0.10±0.10) mA (n=16), and the average delay of wave Ⅲ latency was (0.15±0.21) ms (n=16). Histological morphology analysis showed that the contact surface of cochlear nucleus around the electrode showed vacuolar changes at 48 h post-operation, and increased glial components were observed at the edge of DCN at 48 h, 7 d and 180 d post-operation. Results of q-PCR showed IL-1β and MMP9 were upregulated at 48 h and 7 d post-operation compared with the unimplanted electrode group, but there was no significant difference.

Conclusion·The modeling success rate of established ABI-implanted guinea pig model is high and have outstanding repeativeness, and the local gliosis is preliminarily observed after implantation.

Key words: auditory brainstem implantation (ABI), animal model, guinea pig, auditory electrophysiology, histological morphology

中图分类号:

R764.4

周祥, 潘金锡, 张钦杰, 李蕴, 陈颖, 谭皓月, 彭飞, 黄穗, 谭治平, 吴皓, 贾欢. 听觉脑干植入豚鼠模型构建的标准化步骤及评价[J]. 上海交通大学学报（医学版）, 2022, 42(5): 583-590.

ZHOU Xiang, PAN Jinxi, ZHANG Qinjie, LI Yun, CHEN Ying, TAN Haoyue, PENG Fei, HUANG Sui, TAN Zhiping, WU Hao, JIA Huan. Establishment and evaluation of standardized steps for building a guinea pig model of auditory brainstem implantation[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(5): 583-590.

图/表 6

图1 豚鼠ABI手术流程Note：A. The black dotted line in the figure shows the scope of skin incision. B. Exposing the skull and grinding out the scope of auditory vesicles (The black dashed line shows the area of removed bulla). C. Position relationship between cochlea and meningeal incision (The white arrow shows round window of cochlea. The black arrow shows the pia mater). D. DCN surface in the visible state (The black dotted line shows the actual visible DCN range during the operation).

Fig 1 ABI surgical procedure in guinea pigs

图2 豚鼠专用ABI外形及电极局部示意图Note：The arrows indicate the electrode contacts and the corresponding connectors.

Fig 2 Schematic diagram of ABI electrode for guinea pig

图3 术中及术后eABR波形图Note：A. eABR test was performed at cochlea intraoperatively, and waves Ⅰ and Ⅳ on the operated side were well observed. B/C. eABR waveforms were elicited from the surface of DCN intraoperatively (B) and postoperatively (C), and wave Ⅰ, Ⅱ and Ⅲ on the operated side were well observed.

Fig 3 eABR waveform elicited intraoperatively and postoperatively

图4 术后双侧耳蜗核及脑干组织H-E染色Note：A. Partial view of the unimplanted side corresponding to the black square in Figure C. B.Partial view of the implanted side corresponding to the red square in Figure C. C. H-E staining of bilateral cochlear nuclei and brainstem tissues. The blue dotted line area is DCN.

Fig 4 H-E staining of bilateral cochlear nuclei and brainstem tissues after operation

图5 术后DCN免疫荧光染色图Note：White arrows indicate areas of increased astrocytes and microglia. The star shows that the cochlear nucleus is separated from the brain stem region.

Fig 5 Immunofluorescence staining of postoperative DCN

图6 q-PCR检测豚鼠电极植入后DCN部位 IL-1β 和 MMP-9 mRNA表达量

Fig 6 mRNA expression of IL-1β and MMP-9 in DCN after implantation in guinea pigs by q-PCR

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听觉脑干植入豚鼠模型构建的标准化步骤及评价

Establishment and evaluation of standardized steps for building a guinea pig model of auditory brainstem implantation

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