这项工作得到了来自 NIDCD 司内研究/NIH (DC000082-02 到 W.W.C., 以及 DC000081 先进成像核心) 的资金支持。我们感谢 NIDCD 动物设施的工作人员照顾我们的动物。

所有动物程序均经国家耳聋和其他传播疾病研究所 (NIDCD ASP1378-15) 动物护理和使用委员会批准。
1. 程序设置和准备
<ol><li>实验开始时用环氧乙烷消毒所有仪器。动物之间, 清洁的仪器使用珠杀菌。</li>
	<li>将含有病毒基因治疗的溶液载入微注射器的微中。本研究使用的病毒载体是 AAV2/8-whirlin (每毫升 1 x 1013基因组拷贝, 见材料表)。 注意: 通常, 1.1 µL 总卷加载到微中。</li>
</ol>2. 麻醉
注: 本研究使用的鼠标应变是 whirler 鼠。使用了纯合突变体 (Whrnwi-fi) 和异型窝 (Whrn+/无线)。
<ol><li>把母亲放在一个独立的笼子里 (与垃圾分开)。</li>
	<li>将含有垃圾 (P0 P5 幼崽) 的家庭笼子放在循环热垫上 (设置在37.5 摄氏度) 以保持老鼠的体温。</li>
	<li>切掉一只乳胶手套的拇指部分, 然后在里面放一只小狗。</li>
	<li>将小狗用乳胶手套拇指放入一桶冰中2分钟。</li>
	<li>将麻醉的小狗放在一个大广场商业塑料冷冻包装上, 幼崽和包表面之间有 4 "x 4" 纱布。</li>
	<li>用碎冰填充一只重型乳胶手套, 并将冰手套放在幼崽周围。</li>
	<li>检查小狗是否有充分的麻醉, 完全缺乏对各种刺激的反应 (包括坚定的脚趾捏)。在手术期间把幼崽留在冰袋上 (大约 5-10 分钟)。 注意: 我们建议在手术期间将幼崽放在冰包上不超过15分钟。</li>
</ol>3. 手术方法 (图 1)
<ol><li>当动物被麻醉后, 用碘擦拭和酒精擦拭来清洁耳后的皮肤。</li>
	<li>用微剪刀在耳后2毫米耳后切口, 用微剪刀将胸锁乳突肌分开。</li>
	<li>识别脸部神经和泡。泡在这个年龄是软骨和半透明的, 它位于面神经的内侧。stapedial 动脉可以通过泡在这个年龄看到, 这是一个有用的里程碑。</li>
	<li>遵循面部神经优和向后定位后半圆管 (PSCC)。用微剪刀去除后半规管上的肌纤维和软组织。 注意: PSCC 在这个年龄是软骨的。</li>
	<li>在微喷油器上使用玻璃微 (直径10微米) 穿透 PSCC。</li>
	<li>将病毒基因治疗注入内耳。 注: 通常, 20 注射 49 nL 的基因治疗被交付到后半圆运河超过四十年代 (总容量 ~ 1 µL)。使用的病毒滴度为每毫升 1 x 1013基因组拷贝。</li>
	<li>用 5-0 polyglactin 缝合闭合皮肤切口。</li>
</ol>4. 术后护理
<ol><li>将小狗放在暖垫上, 以恢复从麻醉中恢复正常体温, 经常手动刺激/滚动与戴手套的人的手指。</li>
	<li>一旦小狗醒了, 把它放回它的笼子里。</li>
	<li>爱抚每一个小狗, 棉花拭子已经暴露在家庭笼床上用品。 注意: 这样做的目的是让老鼠的气味, 因为他们在手术前, 这增加了母亲再次接受她的垃圾后手术的可能性。如果可能的话, 母亲的尿液可以用棉签来收集和揉在幼崽身上, 以进一步降低拒绝的可能性。</li>
	<li>将矿物油应用到母亲的鼻子上, 脱敏她的 8, 并将母亲重新引入家庭笼子.</li>
</ol>

<ol>
	<li>Chien, W. W., Monzack, E. L., McDougald, D. S., Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+sensorineural+hearing+loss.">Gene therapy for sensorineural hearing loss.</a> Ear Hear. 36, 1-7 (2015).</li><li>Askew, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tmc+gene+therapy+restores+auditory+function+in+deaf+mice.">Tmc gene therapy restores auditory function in deaf mice.</a> Sci Transl Med. 7, 295 (2015).</li><li>Akil, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75, 283-293 (2012).</li><li>Pan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+restores+auditory+and+vestibular+function+in+a+mouse+model+of+Usher+syndrome+type+1c.">Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c.</a> Nat Biotechnol. 35, 264-272 (2017).</li><li>Isgrig, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+Restores+Balance+and+Auditory+Functions+in+a+Mouse+Model+of+Usher+Syndrome.">Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.</a> Mol Ther. 25, 780-791 (2017).</li><li>Lentz, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+hearing+and+vestibular+function+by+antisense+oligonucleotides+in+a+mouse+model+of+human+deafness.">Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.</a> Nat Med. 19, 345-350 (2013).</li><li>Shibata, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+Prevents+Autosomal-Dominant+Hearing+Loss.">RNA Interference Prevents Autosomal-Dominant Hearing Loss.</a> Am J Hum Genet. 98, 1101-1113 (2016).</li><li>Van Sluyters, R. C., Obernier, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+care+and+use+of+mammals+in+neuroscience+and+behavioral+research.">Guidelines for the care and use of mammals in neuroscience and behavioral research.</a> Contemp Top Lab Anim. 43, 48 (2004).</li><li>Chien, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+Restores+Hair+Cell+Stereocilia+Morphology+in+Inner+Ears+of+Deaf+Whirler+Mice.">Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.</a> Mol Ther. 24, 17-25 (2016).</li><li>Hirose, K., Hartsock, J. J., Johnson, S., Santi, P., Salt, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+lipopolysaccharide+compromises+the+blood-labyrinth+barrier+and+increases+entry+of+serum+fluorescein+into+the+perilymph.">Systemic lipopolysaccharide compromises the blood-labyrinth barrier and increases entry of serum fluorescein into the perilymph.</a> J Assoc Res Otolaryngol. 15, 707-719 (2014).</li><li>Okada, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+targeting+mouse+vestibule+using+adenovirus+and+adeno-associated+virus+vectors.">Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors.</a> Otol Neurotol. 33, 655-659 (2012).</li><li>Suzuki, J., Hashimoto, K., Xiao, R., Vandenberghe, L. H., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy+with+ancestral+AAV+in+adult+mice:+complete+transduction+of+inner+hair+cells+without+cochlear+dysfunction.">Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction.</a> Sci Rep. 7, 45524 (2017).</li></ol>

作者没有相关的披露。

几种手术方法被描述为进入啮齿动物内耳。Cochleostomy 和圆形窗口的方法是最常用的进入耳蜗, 而后半圆管和淋巴囊方法通常用于访问前庭器官1。在最近的一项研究中, 我们发现, 后半规管注射的病毒基因治疗导致高效率的头发细胞转导的前庭器官和耳蜗5。事实上, 耳蜗毛细胞转导是通过后半圆管注射更高, 当与圆形窗口注射相比, 同一鼠遗传性听力损失和前庭功能障碍的模型5,9。考虑到圆窗与耳蜗的解剖距离, 与后半圆管注射相比, 圆窗注射可能导致耳蜗毛细胞转导下降, 这似乎自相矛盾。这一发现可以解释为一个事实, 即圆形窗口位于靠近耳蜗渡槽。因此, 当病毒基因治疗通过圆形窗口注射时, 其浓度可能会被来自耳蜗渡槽10的脑脊液稀释。
其他研究报告11,12, 发现后半圆管注射液导致耳蜗和前庭毛细胞转导。在 AAV 的研究中, 报道了耳蜗和前庭毛细胞的转导. 但是, 没有进行数值量化。铃木 (AAV-Anc80-GFP) 的研究表明, 在成年小鼠的后半规管方法中, 有高水平的耳蜗和前庭毛细胞转导. 在成年小鼠中, 由于后半圆管的骨覆盖完全僵化,12, 采用小导管插管是比较可取的。成年小鼠后半规管插管可减少注射回流, 降低转导效率。这一步骤是不需要在新生小鼠, 因为骨覆盖的后半圆管仍然软骨在那个年龄。
后半圆管注射的缺点之一是, 不能确定注射基因治疗是否交付外淋巴液或 endolymph。尽管有这个缺点, 后半圆运河解剖上很容易定位, 需要对颞骨进行最小的操作来识别。这就减少了因手术外伤而导致内耳损伤的几率。后半圆管注射液是内耳基因治疗分娩的一个很有吸引力的选择。

内耳基因治疗是一个快速发展的研究领域。它已应用于各种动物模型, 以对抗耳毒性, 噪音损伤和遗传性听力损失1。最近的几项研究表明, 在内耳基因治疗后突变小鼠听力和平衡功能的功能恢复2,3,4,5,6,7. 确定内耳基因治疗成功与否的关键因素之一, 是用于进入内耳的手术方法。理想的做法是, 手术方法很容易执行, 解剖标志将是一致的, 易于辨认, 并由此产生的定向细胞类型的转导是很高的。
在最近的一项研究中, 我们表明, 当病毒基因治疗是通过 whirler 突变小鼠的后半规管注射 (一种听觉丧失和前庭功能障碍模型), 在前庭中看到的感觉毛细胞的有效传导器官并且在耳蜗5。感觉毛细胞转导的高效性导致了这些突变小鼠听觉和前庭功能的改善。
在本文中, 我们详细描述了后半圆管的方法, 以获得新生小鼠内耳。

<table border="0" cellpadding="0" cellspacing="0" style="width:735px;" width="733">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Operating microscope</td>
			<td style="width:113px;">Zeiss</td>
			<td style="width:111px;"></td>
			<td style="width:295px;">OPMI Pico ENT microscope. Other dissection microscopes would also work.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro-forcepts</td>
			<td style="width:113px;">Fine Science Tools</td>
			<td style="width:111px;">11251-10, 11295-51</td>
			<td>#5 and #55 Dumont</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro-scissors</td>
			<td style="width:113px;">Fine Science Tools</td>
			<td style="width:111px;">15002-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nanoliter2000 microinjector</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:113px;">Mastex</td>
			<td>Model 500/600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-0 vicryl sutures</td>
			<td style="width:113px;">Ethicon</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AAV8-whirlin</td>
			<td style="width:113px;">Vector Biolabs</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass pipette</td>
			<td style="width:113px;">Sutter Instruments</td>
			<td style="width:111px;">B100-75-10</td>
			<td>Borosilicate glass</td>
		</tr>
	</tbody>
</table>

posterior semicircular canal approach for inner ear gene delivery in neonatal mouse

内耳基因治疗提供了一个伟大的承诺, 作为一种潜在的治疗听力损失和头晕。内耳基因治疗成功的关键决定因素之一是找到一种分娩方法, 从而使靶细胞类型的传导效率保持在最小, 同时最大限度地减少听力损失。在本研究中, 我们描述了后半规管方法作为一种可行的方法, 为内耳基因分娩的新生小鼠。我们表明, 通过后半规管的基因传递能够灌注整个内耳。简单的解剖鉴定的后半圆管, 以及最低限度的操作所需的颞骨, 使这种手术方法是一个有吸引力的选择内耳基因传递。

注射 AAV8-whirlin 基因治疗新生儿 whirler 小鼠通过后半规管导致 whirlin 表达 (绿色) 在 utricular 毛细胞 (图 2), 整体感染效率为 53.1% (SD 38.1, n = 28)5.转基因毛细胞有拉长的张图片 (红色) 与对侧非注射耳朵的毛细胞 (5.35 @ 2.11 µm vs 3.20 @ 0.34 µm, 分别) 5.
AAV8-whirlin 后半圆管注射液也导致 whirler 小鼠耳蜗毛细胞的转导 (图 3)。平均内毛细胞感染效率为 77.1% (SD 12.7, n = 8)5。转基因毛细胞表达 whirlin (绿色) 在张图片提示和有拉长张图片 (红色) 相比, 从对侧非注射耳朵的毛细胞 (5.04) 0.72 µm vs 1.01 @ 0.08 µm 在耳蜗顶点, 分别)5。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56648/56648fig1.jpg"> 图 1: 术中图像. 术中图像显示 P0 小鼠手术进入后半圆管 (PSCC)。显示左耳。PSCC 用虚线黑线勾勒出来。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56648/56648fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56648/56648fig2.jpg"> 图 2: 前庭毛细胞转基因使用 PSCC 基因传递. AAV8-whirlin 通过 PSCC 方法交付, 导致 utricular 毛细胞感染的高水平。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56648/56648fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56648/56648fig3.jpg"> 图 3: 耳蜗毛细胞转基因使用 PSCC 基因传递. AAV8-whirlin 通过 PSCC 的方法, 导致了高水平的耳蜗毛细胞感染。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56648/56648fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse at JoVE.com

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

在本研究中, 我们描述了后半圆管方法作为一种可靠的方法, 为内耳基因分娩的新生小鼠。我们表明, 通过后半规管的基因传递能够灌注整个内耳。

后半圆管法在新生小鼠内耳基因传递中的研究

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

posterior-semicircular-canal-approach-for-inner-ear-gene-delivery

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K 次观看.  National Institutes of Health. 在本研究中, 我们描述了后半圆管方法作为一种可靠的方法, 为内耳基因分娩的新生小鼠。我们表明, 通过后半规管的基因传递能够灌注整个内耳。

视频: Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

科研

医学

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

基于音频的虚拟游戏环境的发展，以帮助盲人的导航技巧

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

异位黏膜移植程序直接给药到小鼠的大脑

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

一种非人类灵长类常见狨中内耳药物的外科手术方法 (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Posterior Approach for Debridement of the Psoas Abscess

This method focuses on outlining a safe zone for irrigation and debridement of a psoas abscess through a posterior approach. Initially, an anterior approach to the spine was performed to ensure that the anterior longitudinal ligament and the psoas muscle could be visualized. All the abdominal organs were removed. Subsequently, a posterior approach was performed to remove the paraspinal muscles from L1&#8211;L5. The transverse processes, pars interarticularis and lamina of L1&#8211;L5 were identified. The exiting nerve root was identified between the transverse processes and followed into the substance of the psoas muscle. Using the anterior and posterior approach, the lumbar plexus was isolated from the substance of the psoas muscle. Before and after various steps of dissection, digital photographs were obtained. These images were uploaded into ImageJ and multiple measurements, including the distance between the lateral superior and inferior tip of each TP to the most lateral region of the plexus, the distance between the lateral superior and inferior tip of the TP to the lateral edge of the psoas, and the width of the lumbar plexus were recorded. The safe zone for entering the substance of the psoas muscle was defined between the lateral edge of the psoas muscle and the lateral edge of the lumbar plexus. The relationship of this interval to the tip of the transverse process at each level was measured and reported.

This is a cadaveric study investigating the landmarks for the posterior approach for irrigation and debridement of the psoas abscess. The interval between the transverse processes (TP) was used to access the substance of the psoas muscle.

切除普索阿脓肿的后向方法

This method focuses on outlining a safe zone for irrigation and debridement of a psoas abscess through a posterior approach. Initially, an anterior ...