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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

摘要

调查使用病毒介导的基因转移到阻止或逆转听力丧失在很大程度上被转移到外周听觉系统。很少有研究探讨基因转移到中央听觉系统。脑干的背侧耳蜗核(DCN),它包含了听觉通路的二阶神经元,是用于基因转移的潜在位点。在这个协议中,对于鼠DCN经由后颅窝的方法直接和最大曝光技术被证实。这种方法允许对急性或存活手术。继DCN的直接可视化,一台主机的实验是可能的,包括注射视蛋白进入耳蜗核和随后刺激的由光纤耦合到蓝色光的激光。其他神经生理学实验,如电刺激和神经注射器描也是可行的。visualiza的水平化和刺激的持续时间可达到使这种方法适用于广泛的实验。

引言

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam®2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9

Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14

Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20

The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

研究方案

注:所有的实验程序按照马萨诸塞州眼的动物护理和使用委员会及耳科医院和哈佛医学院,这遵​​循国家动物保健指南,包括公共卫生服务政策上的人文关怀和使用实验动物,在执行ILAR指南,以及动物福利法。上市实验步骤如下左DCN的细节曝光。使用无菌器械,同时执行的生存术。

1.主开颅手术和背耳蜗核曝光

  1. 麻醉
    1. 通过腹腔内给药麻醉小鼠,年龄8-12周,体重18-24克,用甲苯噻嗪20mg / kg的氯胺酮和100毫克/公斤。确定合适的麻醉监测心脏,呼吸率,以及脚趾捏撤回反射。对眼睛的地方兽医软膏,以防止干燥,而在麻醉下。一旦充分麻醉下,头发剃覆吨他头皮以提供无障碍地访问手术部位。
  2. 手术定位
    1. 将鼠标牢固在小动物立体定位架,由一吻夹具保持在适当位置。
    2. 确保吻钳位足够松,以便有足够的呼吸,但不够紧完全固定在鼠标的头部。如果鼠标头是松散的,动物是可能的过程的开颅部分期间掉出的头支架。
    3. 放置在标准的方式,这允许心脏速率监测听觉脑干植入电极。呼吸频率应该由可视化监控。需要注意的是正常的心脏速率和呼吸速率会根据鼠标的年龄而变化。
    4. 温度计放置和euthermia确保通过放置在恒温加热毯。
  3. 切开皮肤及颅骨interparietal和枕骨骨曝光
    1. 下一个microscOPE,使透过皮肤的垂直切口处开始的中线,耳廓直接之间,并延伸到后头部的尾部。下面正中切开皮肤,横向位移皮肤。可视覆盖颅骨的左后侧方面的肌肉。
    2. 肌肉附件离断取下的肌肉覆左顶叶,interparietal和颅骨枕骨骨各自的骨头用手术刀或虹膜剪刀。观察一个小的程度以及切割边缘的肌肉出血。通过用棉签10-15秒温和的压力减少了。
  4. 开颅覆耳蜗核
    1. 确定相关缝合线,包括矢状面和lambda缝合线( 图1,左图)。
    2. 用咬骨钳,作开颅手术在interparietal骨,中线左侧,〜2毫米尾鳍在lambda缝合线。这个区域覆盖了DCN。
    3. 跟随着克开颅手术,观察薄层硬膜覆盖在小脑。使用手术刀,删除硬脑膜。 ( 图1,中图)。
      注:去除硬脑膜可能导致小程度的出血。使用手术刀刀片去除硬脑膜的这个过程是为了减少小脑抽吸,这将集中在脑干表面和模糊的识别与DCN期间血液损失量。小鼠总血量为〜1.5毫升不应超过>低于15%。如果出血超过这个数额,鼠标应提供与补液。
    4. 取出凝固的血液覆盖的棉签涂抹小脑。或者,用牙科点到小脑清除血液离开血淌轻轻生理盐水。
  5. 小脑吸
    1. 使用5法语吸,吸左侧小脑覆DCN的最外侧部分,直到与DCN是相UAL( 图1,右图 )。卸下左侧小脑大约1/4到1/3。毗邻DCN的主要地标是上半规管的壶腹。
      注:小脑心愿是协议的关键一步。如果它发生在一个单一的企图,而不是与吸多遍,因为这会导致出血是最成功的进行了小脑导演的愿望。以协助抽吸,设置显微镜的焦平面处,CN,这将是略微远端小脑的表面的期望的深度。焦平面设置到CN将改进的可视化,并确保一个鲜明的图​​像。
    2. 下面愿望,再出血,脑脊液(CSF)集结起来,小脑排量的DCN预期。灌输0.5毫升的无菌盐水的迅速进入开颅以防止血液凝固。然后轻柔涂抹的用牙科点和吸入的组合可以是使用扫清了DCN直接可视化的路径。不要直接接触DCN的表面。

2.压力显微注射的病毒介导的基因转移和手术恢复

  1. 与DCN是免费的覆盖血液和脑脊液后和清晰可见,用10微升Hamilton注射器在2分钟内使压力显微注射入DCN。
  2. 用显微操作引入针,直到针尖不再与DCN的表面下可见。
  3. 为了获得最佳性能,使用33或34号针(使用气密注射器用34号针头)与浅斜面,如45°,以减少钝伤和本地化DCN内注射量,这是一种薄和浅脑干结构(<300微米厚)。
    注意:其他注射手段可以基于利用。理想情况下,注射仪器应具有灵活性,允许一些轻微的奔D,如果需要直接针对DCN。另外,作为小鼠轻微移动整个手术过程中,由于呼吸,仪器应是坚固足以承受运动的少量。

3.手术恢复

  1. 紧接着注射,重新接近皮肤,让鼠标按标准恢复过程来恢复。其余小脑和疤痕组织将填补引起小脑愿望腔。不断地监测动物,直到足够的意识恢复,保持胸骨斜卧。监测心脏速率,呼吸速率,以及饲料的能力。
    注:没有动物已经动过手术返回到与其他动物的笼子,直到完全康复。
  2. 如果鼠标开始走在圈子手术后(<5%的病例),皮肤切口关闭后通常2-4小时,立即牺牲鼠标,因为它很可能有困难的时候喂食。牛逼他的副作用,可能是由于小脑的愿望。动物应为疼痛,术后进行监测,并适当麻醉品应给予保证根据机构的标准动物的舒适性。抗生素可能是必要的,如果感染的证据。

4.二次开颅手术和人工耳蜗核曝光

  1. 2-4周的愈合和病毒介导的基因转移的潜伏期后,重新麻醉与基因转移的类型预先注射小鼠,并重复步骤1.1-1.5孵育时间而变化。
  2. 由镊子,解剖刀,和咬骨钳的组合去除瘢痕组织在开颅站点。
  3. 可视化开颅后,确定剩余的小脑和疤痕组织覆盖在DCN的组合。使用5弗伦奇吸入到吸覆小脑/疤痕组织(类似于前述小脑抽吸)。
  4. 下面的心愿,期待再出血,脑脊液Build镜头,和小脑的其余位。快速引入盐水进开颅以防止血液凝固。用温和的牙齿涂抹点和吸力的组合来清除的DCN直接可视化的路径。
  5. 观察到DCN的表面上。执行光遗传学基础生理学实验为DCN现在访问。引入光刺激,例如用光纤( 图2),以驱动一个光遗传学系的实验。

5.组织学

  1. 下面的实验结论,安乐死鼠标氯胺酮的过量。灌注小鼠生理盐水后跟4%多聚甲醛。
  2. 提取的头骨和后固定2小时脑干。 Cryoprotect在30%蔗糖的脑干中24-48小时。使用一节使用60微米的部分标准的低温​​恒温器脑干。

结果

部分小脑吸入演示进入耳蜗核

后的皮肤和肌肉覆盖在颅骨被除去,颅骨表面的地标,例如冠状和LAMDA缝合线,表明开颅的大致定位。下面开颅咬骨钳,小脑可视化。小脑的小部分仔细吸出表明该CN,然后可将其注入( 图1)的可视化。

二级耳蜗核暴露的刺激与蓝光激光器

继DCN,与视蛋白的基因转移,和潜伏...

讨论

本文介绍了DCN在操纵中枢听觉系统的小鼠模型直接可视化的技术。直接可视化的概括方法提供了主要的替代显著的优势,这是立体的方法。为主,DCN的直接可视化允许对脑干部位的立即确认,而立体定向的方法不能提供直接可视化。在于必要延长潜伏期,如在病毒介导的基因转移的情况下的实验中,存在用于低感染效率的潜力,如果注射液"射门"时,目标位置。另外,直接可视化的方法允许用于?...

披露声明

作者什么都没有透露。

致谢

资金来源:这项工作是由基金会资助贝尔塔雷利(DJL),一个MED-EL补助(DJL),以及健康教育资助DC01089国家研究院​​(MCB)的支持。

材料

NameCompanyCatalog NumberComments
Stereotaxic holderStoelting51500
Homeothermic blanketHarvard507214
Scalpel blade #11Fine Surgical Tools10011-00
Iris scissorFine Surgical Tools14084-08
5 French suctionSymmetry Surgical2777914
Dental PointsHenry Schein100-8170
Bone rongeurFine Surgical Tools16020-14
10 µl Hamilton syringeHamilton 7633-01
34 gauge, needleHamilton 207434
RongeursFine Surgical Tools16021-14

参考文献

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  20. Taberner, A. M., Liberman, M. C. Response properties of single auditory nerve fibers in the mouse. J Neurophysiol. 93 (1), 557-569 (2005).
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