这项工作得到了日本政府下个 KAKENHI (24592560、15H04991 和 15K15624) 和武田科学基金会中频和 Jikei 大学战略优先研究基金中频的资助, 以 H.J.O。

所有实验程序都是在日本东京的 Jikei 大学进行的。动物处理和实验程序经 Jikei 大学机构动物保育和使用委员会批准 (批准号: 26-060), 并按照《 Jikei 实验室动物使用机构指南》执行。大学, 同意由日本科学理事会正确进行动物实验的指南 (2006)。动物护理是按照《护理和使用实验动物指南》 (1996 实验室动物资源研究所) 的建议进行的。
1. 术前检查
<ol><li>采取颅内计算机断层扫描 (CT) 对动物 (常见的猴) 进行检查, 以方便在手术前至少一个月内鉴定个别解剖结构。</li>
	<li>在手术前至少一个月进行听力测试。 注意: 在这里, 我们使用 ABR 来评估听力状态。简要地, 一个正极被皮下放置在顶点和一个参考负极在被刺激的耳朵的背底。地面电极被皮下放置在颈部的背部。17,18 ABRs 以音调爆裂 (1.0 毫秒音爆发持续时间0.1 毫秒上升和下降) 诱发。然后从其最大振幅的10分贝步骤中降低了声音电平。在每个声音水平 , 512 反应是平均的 ( 以刺激极替 ) 。ABR 阈值被定义为最低的声压级 (SPL), 在那里可以检测到任何波。</li>
</ol>2. 麻醉
<ol><li>快速动物从夜间手术前全身麻醉。</li>
	<li>管理4% 异氟醚和环境空气在 5-6 毫升/分钟与掩模到面通风或在感应室为慢感应 (图 1A)。确认动物不响应疼痛刺激, 如脚趾捏。</li>
	<li>Anesthestize 常见的猴肌肉注射 (入股肌) 的组合 medetomidine (0.04 毫克/千克), 咪达唑仑 (0.4 毫克/千克), 和布托啡诺 (0.4 毫克/千克)。</li>
	<li>注射氨苄西林 (0.08 毫克/克) 皮下30分钟或以上皮肤切口前抗生素。</li>
	<li>将狨放在仰卧位, 头部朝向顶部, 伸展头部和颈部。用尼龙线固定上颌骨, 拉起舌头以获得良好的视野, 或者使用喉镜可视化会厌和喉的进入 (图 1B)。</li>
	<li>在会厌的喉表面插入气管导管。 注: 有可能使用16口径静脉注射导管, 或 6-法国标准喂养管, 作为气管插管 (图 1C)。</li>
	<li>检查 EtCO2 , 从气管插管, 以确认它已正确放置到气管。在导管定位和呼吸气体运动检查后, 用胶带将气管插管固定在狨的脸上。</li>
	<li>在手术期间保持1-3% 异氟醚之间的麻醉。</li>
	<li>在操作期间监视 EtCO2、2、体温和直肠温度。在加热垫上执行所有步骤, 防止体温降低。</li>
</ol>3. 术前准备
<ol><li>在仰卧位置准备狨, 并将头部倾斜至 30-45 度角至非手术侧 (图 2A)。</li>
	<li>剃须后耳区的手术侧。</li>
	<li>对手术部位进行酒精和聚维酮碘的消毒, 无论是在耳后区还是外耳道。</li>
	<li>在狨上放置一个带圆孔的外科悬垂板 (图 2B)。</li>
</ol>4. 手术程序
<ol><li>注射1% 盐酸利多卡因 (1:100,000 肾上腺素, 0.1-0.2 毫升) 皮下进入皮肤切口部位。用15号圆形刀片在耳后皮肤上做切口。</li>
	<li>止血时, 用纱布浸泡在 1:5, 000 稀释肾上腺素中, 对出血区施加压力。双极性灼烧也是有效的 (图 2C)。</li>
	<li>打开后耳区软组织, 使耳软骨 (c) 和耳后肌 (m) 可以识别。暴露出应在软骨深处可见的外部听觉管骨 (图 3A)。</li>
	<li>为确定鼓膜的深度, 暴露鼓膜边缘的骨部, 可识别为后耳道最深部的皮肤和骨粘连。 注意: 这一步允许我们认识到中耳腔的侧部有多深 (图 3A)。</li>
	<li>将耳后肌 (胸锁乳突肌、splenius 头癣肌、二腹肌肌后腹部) 剥离至尾部, 与颞骨分离。然后暴露颞骨的侧向表面 (星号在图 3B中)。</li>
	<li>用1.0 毫米的钻石 (图 3C) 在外部听觉管道后面打一个洞, 大约5毫米。使用 1000-8000 rpm 的钻速取决于钻孔位置。 注意: 一个缓慢的速度是足够的微妙操作附近的半圆形运河或面神经。适当滴盐水在钻孔部位, 防止热损伤。</li>
	<li>通过钻孔和确认水平半圆管 (HSC) 的位置来扩大孔, 向乳突腔 (图 3D) 凸出。</li>
	<li>确定垂直部位的面神经在 HSC 前的位置。如果他们存在, 钻和删除所有的骨间隔, 以获得一个清晰的看法, 面部凹进处, 这是位于三角区中心的面神经和鼓索。</li>
	<li>在脸部神经的垂直部分和鼓膜边缘 (图 4A中的星号) 之间钻孔, 并在1000转每分钟0.6 毫米直径的钻石, 使一个小孔。</li>
	<li>将其与后管壁平行移动, 以防止面部神经受损, 通过瘦骨可以看到粉红色线。在将神经暴露为白色线结构之前, 立即停止钻孔 (图 4B)。</li>
	<li>确保圆形窗口利基可以通过孔可视化 (图 4C)。然后, 用25口径或更薄的针头将药物注入利基。为了保留在圆形窗口利基的药物, 在利基 (图 4D) 上放置粘性解决方案。</li>
	<li>在确认止血是安全控制后, 用6-0 根吸收线闭合简单的间断缝合层 (表皮层和真皮层)。</li>
	<li>把纱布固定在手术伤口上, 贴上绷带。</li>
</ol>5. 术后护理
<ol><li>管理盐酸 atipamezole (0.15 毫克/千克) 肌肉入股肌拮抗 medetomidine。</li>
	<li>当自发呼吸开始时, 取出气管导管。</li>
	<li>保持在 30% O2, 29 ℃的重症监护病房的共同狨, 直到麻醉结束, 以完成唤醒。</li>
	<li>确认没有发现提示前庭功能障碍, 如眼球震颤, 并返回动物的饲养笼。</li>
</ol>

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作者没有什么可透露的。

耳蜗血流量非常小, 估计是在人类总心脏输出的 1/100万的顺序, 和访问是有限的血-耳蜗屏障的存在, 分离内耳从一般循环19 ,20。由于这些原因, 药物或病毒的媒介管理进入内耳是有利的, 不是通过系统性的管理, 而是由直接管理。在小鼠和豚鼠实验中, 报告了各种方法, 如采用耳后入路或腹侧方法的转圆窗、cochleostomy、淋巴囊传递和半圆管方法21, 22,23,24,25,26。
在本研究中, 我们采用后鼓膜置和圆窗管理作为一种潜在的翻译研究方法。后鼓膜置是一个成熟的方法为临床手术程序和适用于普通的猴, 因为鼓膜的解剖特征类似于人类, 而其路径乳突主要由一个大单腔有非常薄的骨间隔。事实上, 我们执行了所有的后鼓膜置程序, 没有任何并发症或听力损失的共同狨。如果后鼓膜置的执行, 药物管理可以在明确的看法周围的重要组织, 如半圆形运河, 面神经, 镫和圆形窗口利基。值得注意的是, 通过这种单一的方法, 我们可以通过耳蜗、HSC、后半圆管和圆形窗口利基来管理溶液。根据使用小鼠的研究, 圆窗管理是更少的侵入性, 在耳蜗外淋巴液中提供了比cochleostomy27 更均匀的解决方案扩展。据报道, 直接注射腺病毒通过半圆管基因转移显示基因转导主要在前庭器官26。淋巴囊传递提供直接注射到 endlymphatic 空间, 并报告为病毒载体达到前庭端器官和耳蜗25。然而, 关于效率的信息似乎不够。
为了进行安全的手术, 解剖信息是至关重要的。比较人与普通猴的关系, 中耳鼓膜和内耳的基本解剖特征是相似的。在中耳腔与乳突腔、听骨位置、面神经和鼓索的位置等方面进行手术的重要解剖部位与人完全相似。但是, 存在一些差异。人乳突腔由许多空气细胞组成, 我们需要钻这些细胞来接近内耳。这在临床手术过程中称为 "根治", 需要时间和破坏原形态。另一方面, 常见的狨乳突腔几乎是一个单一的腔与非常薄的骨间隔, 半圆形运河更大, 拱出进入乳突腔与人体解剖相比。这些差异有利于内耳药物的管理。当直接对 endolymph 管理药物时, 可以通过向 HSC 发出一个小窗口, 如以前在其他物种26中所报告的那样, 半圆形运河的方法是可能的。尽管此方法没有在本实验中执行, 但由于其大小和易于探测性, 普通狨的 HSC 可以很容易地执行。然而, 在采取这种方法之前, 应考虑 lymphorrhea 的风险, 这可能诱发听力障碍。我们认为半圆管方法不是一个理想的实验系统, 用来评估手术后的听力水平。一般而言, 为了防止 lymphorrhea, 用脂肪组织和纤维蛋白胶包装窗户是有效的。
在这种情况下, 我们把重点放在一个较小的侵入性手术, 并放置解决方案的圆形窗膜无穿刺。然而, 单一的管理似乎没有有效的提供足够剂量的药物或病毒载体的外淋巴液。解决这一问题的一个简单方法是在圆窗膜上制造一个孔, 并直接注入溶液。如上文所述, 在内耳形成一个洞会导致 lymphorrhea, 这可能会诱发人的听力丧失和前庭功能障碍, 所以这将很难应用于临床设置。另外, 与啮齿目动物不同的是, 绒猴最初住在树上, 在饲养笼中移动立体感, 因此前庭功能障碍也可能是一个关键的并发症。因此, 在不穿透圆窗膜的情况下, 提高药物扩散效率将是今后的重要内容。一种可能性是使用渗透泵和持续的准备。渗透泵通过导管固定在圆窗膜上, 提供连续的管理。目前, ploxamer407 和明胶水凝胶可作为圆窗膜药物的持续制备。这些设备和载体已经用于临床试验28,29,30,31。
简而言之, 这种手术方法使我们能够在灵长类动物体内对内耳进行安全准确的药物管理, 这对于获得转化研究概念的临床前证明具有重要意义。

感音神经性听力丧失 (SNHL) 主要出现在耳蜗损伤或缺陷。SNHL 的常见原因包括老化 (例如聋)、遗传缺陷、暴露于噪音、感染和毒性药物1。世界卫生组织 (卫生组织) 估计, 占世界人口5.3% 的3.6亿多人患有听力损失2。据估计, 在900至1的2500新生儿中, 有轻度、严重和深刻的先天性永久性听力丧失, 而1/3 岁以上的成年人有一定程度的听力损失65。然而, 对于这些患者来说, 听力丧失没有有效的临床治疗方法。
听力研究一直使用啮齿动物或豚鼠模型, 并提出了许多方法, 如基因治疗和再生治疗, 被认为是一种新的治疗听力损失。然而, 人类和啮齿动物在听觉系统上有很大的区别, 很难将动物模型转化为人类的应用。狨 (jacchus) 是来自亚马逊的新一代世界猴, 由于多种原因, 它已成为各种基础研究研究的一种具有吸引力的非人类灵长类动物模型。首先, 它的解剖和生理学更类似于人类而不是啮齿动物。第二, 关于这个物种的基本生物学信息, 包括疾病、神经网络、行为和基因组, 都有很好的特点。听觉和声带处理, 周期性皮质编码, 音高的表示, 和常见的猴的听觉声音互动也被报告了4,5,6,7,8,9,10. 在最近的组织学研究中, 研究人员发现了20种耳聋基因和阴离子交换器在普通狨耳蜗中的独特表达模式, 发现五基因是渐进性耳聋的病因, 三阴离子交换器, 有不同的表达模式, 从啮齿动物11,12。这些概况使我们相信, 共同狨是听觉研究的有力工具。
作为实验动物的常见狨的最显著属性如下:
<ul><li>简单的处理与小体型相比, 一些老世界的猴子: 成年猕猴重 300-400 克, 身高约60厘米, 这是类似于大鼠。</li>
	<li>高度繁殖的灵长类动物: 在18月的年龄, 它能达到性成熟, 每年能承受两次, 每年产生4到6个子代。</li>
	<li>基因修改是可利用的: et 等人成功地创建了转基因的13和敲出14灵长类动物使用猴的基因修饰涉及神经系统疾病 (例如,帕金森的疾病和阿尔茨海默病)。</li>
	<li>胚胎干细胞和诱导的多能干细胞 (iPS) 细胞已经建立了15,16。虽然与啮齿目动物相比, 保持相同数量的绒猴是相当困难的, 但干细胞或 iPS 细胞的可用性提供了体外化验, 这将减少所需的体内化验的数量。</li>
</ul>为了促进耳聋及其潜在治疗领域的更好的翻译研究, 我们建立了一种利用听觉脑干反应 (ABRs) 进行 CT 和 MRI、全身麻醉和听力测试的成像研究协议。这些实验系统可以为我们提供更好的机会, 以获得临床前证明的概念研究, 这对于弥合啮齿类动物研究和临床试验之间的差距是必不可少的。在这里, 我们报告一个外科方法, 通过圆窗膜对普通狨的内耳进行药物管理。为了在圆形窗口周围获得清晰的视野, 在不操作可能导致传导性听力损失的鼓膜的情况下, 从乳突腔中接近是有用的。临床上, 这种方法, 称为 "后鼓膜置" 是很好的建立, 通常用于人工耳蜗植入和胆脂瘤手术。我们相信, 后鼓膜置允许我们进行准确的药物管理, 而不诱发听力损失。

<table border="0" cellpadding="0" cellspacing="0" style="width:675px;" width="674">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">common marmoset</td>
			<td style="width:92px;">CLEA Japan</td>
			<td style="width:160px;">EDM:C.Marmoset(Jic)</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane</td>
			<td style="width:92px;">Phizer</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">medetomidine</td>
			<td style="width:92px;">ZENOAQ</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">midazolam</td>
			<td style="width:92px;">astellas</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">butorphanol</td>
			<td style="width:92px;">Meiji Seika</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">ampicillin</td>
			<td style="width:92px;">Meiji Seika</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lidocaine hydrochloride</td>
			<td style="width:92px;">AstraZeneca</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;6-0 absorbent thread</td>
			<td style="width:92px;">ETHICON</td>
			<td style="width:160px;">RD-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">atipamezole hydrochloride</td>
			<td style="width:92px;">ZENOAQ</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">phosphate buffered saline</td>
			<td style="width:92px;">Wako</td>
			<td style="width:160px;">045-29795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surge Wave</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">TR-900-OR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diamond Bar 006</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">14070057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diamond Bar 010</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">14070081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">intensive care unit</td>
			<td style="width:92px;">Menix</td>
			<td style="width:160px;">P-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a surgical procedure for the administration of drugs to the inner ear in a non-human primate common marmoset (callithrix jacchus)

虽然在某些疾病中, 人类的症状不能概括, 但长期以来, 啮齿类动物模型已经促进了听觉研究。常见的狨 (Callithrix jacchus) 是一种小型的、易于处理的新世界猴, 它具有类似于颞骨的解剖, 包括中耳听骨链和内耳对人类, 比与啮齿动物的比较。在这里, 我们报告一个可重现的, 安全的, 合理的手术方法的耳蜗圆形窗口的利基为药物传递到内耳的共同狨。我们采用后鼓膜置, 这是一个在人体外科临床应用的程序, 以避免对鼓膜的操纵, 可能导致传导性听力丧失。这个手术过程并没有导致任何重大的听力损失。这种方法是可能的, 由于共同狨的大泡结构, 虽然侧半圆管和面神经的垂直部分应仔细考虑。这种手术方法使我们能够在无听力损失的情况下对药物进行安全、准确的管理, 这对于获得临床翻译研究概念的前期证明具有重要意义。

术后鼓膜置无并发症, 如手术部位感染, 血管损伤出血, 前庭功能障碍, 有时发生中耳操纵。我们在7岁的普通狨的右耳上进行后鼓膜置, 并将1µL 磷酸盐缓冲盐水用于圆窗利基。ABRs 在手术前和两个月后进行测量 (图 5A-B)。ABRs 的波形和阈值与以前的研究17、18相同, 在手术前后这些参数没有变化 (图 5C)。因此, 我们得出结论, 后鼓膜置是一个安全和有用的程序, 药物管理的圆形窗口利基的共同狨。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56574/56574fig1.jpg"> 图 1: 气管插管.(A) 麻醉与4% 异氟醚和环境空气在5-6 毫升/分钟的面罩对脸通气的共同狨。或者, 将普通狨放入感应腔内。(B)将普通狨置于仰卧位, 头部朝向顶部, 并伸展头部和颈部。将上颌骨用尼龙线固定在桌子上, 然后拉起舌头以可视化会厌 (箭头) 和喉的进入。(C)在会厌的喉表面插入气管导管。16口径静脉导管或 6-法国标准喂养管可作为气管导管使用。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56574/56574fig1large.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/56574/56574fig2.jpg"> 图 2: 皮肤切口.(A) 将普通狨置于仰卧位, 并将头部倾斜至30-45 度角至非手术侧。用聚维酮碘对手术侧后耳区进行剃须消毒。Cr, 颅侧, 钙, 尾侧, L, 左侧, R, 右侧。(B)黑线表示皮肤切口线。在皮肤切口之前, 注射1% 利多卡因 (1:100,000 肾上腺素, 0.1-0.2 毫升) 皮下。(c)切开皮下组织直到耳软骨 (c) 升高。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56574/56574fig2large.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/56574/56574fig3.jpg"> 图 3: 钻进乳突腔。(A)切除软组织后, 耳后肌将可见。m, 耳后肌, c, 耳软骨。(B)颞骨表面 (星号) 在耳后肌从骨骼剥离向尾部方向时暴露。pneumatized 结构可以很容易地识别通过颞骨。(C)在外部听觉管骨后, 用1.0 毫米的钻石在大约5毫米的钻孔中钻出了一个孔。(D)常见狨的乳突腔主要由单个腔组成。通过扩大钻孔, 可以很容易地识别水平半圆管 (HSC 和虚线) 向乳突腔隆起。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56574/56574fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56574/56574fig4.jpg"> 图 4: 后鼓膜置。(A)面部神经 (FN) 通过称为 "面管" 的骨通道, 在外耳道骨 (PAC) 和水平半圆管 (HSC) 的后壁之间运行。神经的前区是面部凹进处 (星号)。(B)通过用直径0.6 毫米的菱形条钻入面部凹槽来制造一个孔。(C)通过放大孔, 圆形窗口利基 (RWN) 变得可见。(D)通过面部凹进处的孔, 可以用25口径或更薄的针将溶液 RWN (箭头)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56574/56574fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56574/56574fig5.jpg"> 图 5: 听觉脑干反应 (ABR) 评估.后鼓膜置在一个7岁的普通狨, 和1µL 磷酸盐缓冲盐水被管理到圆形窗口的利基。ABRs 在手术前后测量, 其代表波形分别显示在(A)和(B)中。ABRs 的波形和阈值以六频率的音调爆发而引起, 与以前的研究17、18相似。(C)手术后没有 ABR 阈值的变化。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56574/56574fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus at JoVE.com

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

我们报告一种外科方法, 以管理药物的内耳的非人类灵长类, 常见的狨 (Callithrix jacchus), 通过圆窗膜。

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 ...

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

8.5K 次观看.  Jikei University School of Medicine. 我们报告一种外科方法, 以管理药物的内耳的非人类灵长类, 常见的狨 (Callithrix jacchus), 通过圆窗膜。

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

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

科研

医学

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 ...

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

外科技术治疗脊髓交付:在哥廷根小型猪演示程序

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

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 ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

外科手术切除为鼠标肋骨:模型进行大规模的长骨修复

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型:在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...