该项目的资金来自Colleen M. McDowell博士的可自由支配资金。这项工作部分得到了研究预防失明公司对威斯康星大学麦迪逊分校眼科和视觉科学系的无限制资助的支持。我们感谢Abbot F. Clark博士和Weiming Mao博士对灌注器官培养模型的技术援助。我们感谢狮子会眼科移植与研究所（佛罗里达州坦帕市）为人类供体提供眼睛。

根据《赫尔辛基宣言》的规定，眼睛被用于涉及人体组织的研究。
注意：来自信誉良好的眼库（例如，佛罗里达州坦帕狮子眼科移植研究所）的眼睛在死亡后6-12小时内收获，并检测了供体血清的乙型肝炎，丙型肝炎和人类免疫缺陷病毒1和2。一旦它们被接受，眼睛就会在24小时内被解剖并设置在TAS模型中。排除标准包括任何眼部病变。眼睛不会因年龄、种族或性别而被排除在外。为了确保接收时视网膜的活力，从组织供体中收获视网膜外植体并培养7天和14天（补充图1）。这些视网膜也被解离并在培养物中生长出健康的RGC，7天，RGC标记物染色阳性，多重剪接RNA结合蛋白（RBPMS），以及神经丝中的正神经丝轻链（NEFL）染色（补充图2）。.
1. 设备和用品的准备和灭菌
<ol><li>有关所需耗材的完整列表以及供应商和目录编号，请参阅 材料表 。</li>
	<li>使用前，通过高压灭菌或使用环氧乙烷安瓿对所有设备和器械进行灭菌。</li>
</ol>2. 灌注介质的制备
<ol><li>将1%青霉素链霉素（10，000U / mL青霉素，10，000μg/ mL链霉素在0.85%NaCl中）和1%L-谷氨酰胺（200mM）加入1，000mL高葡萄糖Dulbecco的改性Eagle培养基（DMEM）。</li>
	<li>通过0.22μm过滤器对灌注介质进行灭菌。</li>
</ol>3. 跨层自治系统 （TAS） 设置
<ol><li>设置流入注射器（IOP 和 ICP 储液器）。<ol>
<li>将30 mL灌注培养基（第2部分）加入30 mL注射器中。将 3 向旋塞阀连接到 30 mL 注射器。将 0.22 μm 亲水过滤器连接到 3 通旋塞阀。将 15 G 鲁尔短截线适配器连接到 0.22 μm 亲水过滤器。</li>
		<li>从注射器设置中去除气泡。将管路连接到 15 G 鲁尔短截线适配器。用未通风的通用锁盖关闭旋塞阀的侧端口。重复两次设置。</li>
		<li>将一个注射器标记为通道 1 颅内压 （CH1 ICP），将另一个注射器标记为通道 2 眼内压 （CH2 IOP）。</li>
	</ol>
</li>
	<li>设置流出注射器（IOP 和 ICP 储液器）。<ol>
<li>将 3 通旋塞阀连接到 30 mL 注射器。将 15 G 鲁尔短截线适配器连接到 3 路旋塞阀。将管路连接到 15 G 鲁尔短截线适配器。</li>
		<li>用未通风的通用锁盖关闭旋塞阀的侧端口。重复两次设置。将一个注射器标记为 CH1 ICP，将另一个注射器标记为 CH2 IOP。</li>
	</ol>
</li>
</ol>4. 人类全眼球的制备
注意：如果收到整只眼睛，请按照以下步骤将前段与眼睛的后段分开。如果眼睛被一分为二，请从步骤4.4开始。
<ol><li>将整个眼睛放入聚维酮碘溶液中2分钟。</li>
	<li>在无菌磷酸盐缓冲溶液（PBS）中冲洗眼睛以冲洗掉聚维酮碘。重复2次。</li>
	<li>使用镊子和剪刀从整个眼球中取出附件。将赤道处的眼睛一分为二，以分隔眼睛的前段和后段。</li>
	<li>切除视神经鞘。从后段移除玻璃体房。</li>
	<li>如果需要，从后节修剪额外的巩膜，以确保与IOP（底部）腔室的圆形圆顶良好贴合。使用镊子，确保视网膜均匀地分布在节段的后部。</li>
	<li>IOP（底部）腔室设置<ol>
<li>将人后段置于圆形圆顶上方TAS的IOP（底部）腔室中，视神经朝向顶部。</li>
		<li>使用带有四个螺钉的环氧树脂O形圈密封后段，确保紧密密封。</li>
		<li>将管道插入 IOP（底部）腔室的输入和输出端口。将带有含有介质的管子的 IOP 流入注射器插入 IN 端口，将带管子的空 IOP 流出注射器插入 OUT 端口。</li>
		<li>使用推/拉方法将灌注培养基缓慢注入流入口以填充后眼罩，同时通过流出注射器缓慢地将灌注介质拉出，以去除管路中的任何气泡。一旦IN和OUT管都没有气泡，就停止输注培养基。</li>
		<li>将旋塞阀锁定在关闭位置。从 IOP IN 端口过滤器组件中取出 30 mL 注射器，然后用总共 30 mL 培养基重新填充。将 30 mL 注射器更换到过滤器组件上。</li>
	</ol>
</li>
	<li>
ICP（顶部）腔室设置
	<ol>
<li>将ICP（顶部）腔室/盖子放在后段的背面。确保视神经在顶部腔内。用四个螺钉密封顶部腔室。</li>
		<li>将卡套管插入 ICP（顶部）腔室的输入和输出端口。将带有含有介质的管路的ICP流入注射器插入IN端口，并将带有管子的空ICP流出注射器插入OUT端口。</li>
		<li>轻柔而缓慢地将介质注入IN端口以填充ICP室，并使用推/拉方法从管路中除去气泡。一旦ICP室以及IN和OUT管都没有气泡，就停止注入介质。</li>
		<li>将旋塞阀锁定在关闭位置。从端口过滤器组件中的ICP中取出30 mL注射器，并用总共30 mL培养基重新填充。将 30 mL 注射器更换到过滤器组件上。</li>
	</ol>
</li>
</ol>5. 数据记录系统设置
注：数据记录系统由8通道电源、多通道桥式放大器、静液压传感器和带数据采集软件的计算机组成（见 材料表）。下面介绍如何设置和校准系统。
<ol><li>将电源线连接到 8 通道电源的背面，然后插入备用电池设备。</li>
	<li>将 USB 电缆从 8 通道电源连接到计算机背面。</li>
	<li>使用随附的 I2C 电源线将 8 通道电源连接到多通道桥式放大器。</li>
	<li>将卡口 Neill-Concelman （BNC） 电缆连接到 8 通道电源前面的通道输入，并将电缆末端连接到多通道放大器背面的相应通道。</li>
	<li>将传感器电缆连接到多通道放大器的前面。</li>
	<li>在计算机上安装数据采集软件。<ol>
<li>从随附的软件光盘运行数据采集软件设置安装程序。</li>
		<li>按照计算机屏幕上的说明进行操作。</li>
		<li>安装完成后，选择" 完成"。</li>
	</ol></li>
	<li>打开 8 通道电源。</li>
	<li>打开计算机并启动数据采集软件。<ol>
<li>选择 文件 | 新增功能。
</li>
		<li>选择 设置 | 频道设置。选择三个通道（屏幕左下角）。在 "通道标题" 列中，按如下方式重命名通道：CH1 ICP;CH2 IOP;CH3 TLPG （IOP-ICP）.</li>
		<li>为所有通道上的 范围 选择 2 mV。在 "计算 "列中，为通道 1 和 2 选择" 无计算 "。</li>
		<li>在" 计算 "列中，为通道 3 选择" 算术 "。在 "公式 "部分中：选择 通道/CH2;选择 算术"-";选择 通道/CH1。在" 输出 "部分中，选择" mmHg"。选择 "确定"。再次选择 "确定" 。</li>
	</ol></li>
	<li>设置和校准静液压传感器。 注：在使用以下方法进行实验之前，必须对静水压力传感器进行校准。<ol>
<li>将静液压压力传感器连接到连接到多通道桥式放大器的传感器管路。</li>
		<li>将充满空气的 30 mL 注射器连接到 CH1 （ICP） 压力传感器的侧端口。将血压计连接到 CH1 （ICP） 压力传感器的底部。</li>
		<li>在图表上，查看数据采集软件的页面，通过左键单击采样时间旁边的箭头设置采样速度，然后选择 100。然后右键单击页面的 CH1 （ICP） 区域。</li>
		<li>选择桥式放大器。选择电源过滤器。选择"零"并等待系统归零，注意不要移动压力传感器。</li>
		<li>捏住压力传感器的白色标签，推动空气通过传感器，直到血压计上获得40 mmHg。松开白色标签，取下注射器和血压计。</li>
		<li>在 "单位转换 "页上，选择"减号 （-）"。突出显示最高高原，表示 40 mmHg。单击第 1 点的 箭头 ，然后输入 40。</li>
		<li>突出显示最低平台以指示 0 mmHg。单击第 2 点的 箭头 ，然后输入 0。为设备选择 mmHg。选择 "确定"。</li>
		<li>选择 "确定" （"桥接放大器"页）。对 CH2 （IOP） 重复步骤 9.1–9.7，对最高平台使用 100 mmHg，对最低平台使用 0。</li>
	</ol></li>
	<li>将 TAS/后段单元连接到数据采集系统。<ol>
<li>将TAS/后段单元放入培养箱（37°C，5%CO 2）中。将 ICP 管从 OUT 端口连接到 CH1 （ICP） 压力传感器。</li>
		<li>将 IOP 管从 OUT 端口连接到 CH2 （IOP） 压力传感器。</li>
		<li>将注射器设置（ICP 和 IOP）与介质从 IN 端口连接到环形支架。</li>
		<li>在 "图表视图 "页上，选择" 开始采样"。通过左键单击采样时间旁边的箭头来设置采样速度，然后选择 慢 速和 1 分钟。</li>
		<li>向上或向下调整环上的注射器，以根据协议要求调节ICP和IOP压力。</li>
		<li>通过推拉法每48-72小时对系统中的培养基进行全身补充。</li>
	</ol></li>
</ol>6. 数据检索和分析
<ol><li>在数据采集软件中打开数据文件。</li>
	<li>在 "数据板 "部分中，单击" 多次添加到数据板" 图标。将出现一个新窗口。<ol>
<li>在 "查找使用 "部分中，从下拉菜单中选择" 时间 "。</li>
		<li>在"选择"部分中，从下拉菜单中选择每 1 小时 1 小时。</li>
		<li>在 "单步执行 "部分中，选择" 整个文件"， 然后单击" 添加"。</li>
	</ol>
</li>
	<li>在 "数据板 "部分中，单击" 数据板视图" 图标。突出显示所有数据并复制/粘贴到电子表格中。</li>
	<li>每 24 小时计算 IOP、ICP 和 TLPG 的均值和标准差。使用电子表格程序和图形中的数据透视表选项整理数据。</li>
</ol>7. 免疫组化和后段的苏木精和曙红染色
<ol><li>从TAS模型中取出各个时间点后的后眼节，并在石蜡化之前固定在福尔马林中。</li>
	<li>切除眼睛以产生矢状组织平面。</li>
	<li>用100%二甲苯，95%乙醇，50%乙醇溶液脱蜡石蜡包埋的片段。</li>
	<li>用PBS洗涤载玻片10分钟，并在室温下用封闭缓冲液阻断1小时。</li>
	<li>具有一抗的标记切片：抗胶原IV（细胞外基质（ECM）标记，NB120-6586，1：100）和抗层粘连蛋白（ECM标记，NB300-144，1：100，抗RBPMS（RGC标记），GTX118619，1：50）。</li>
	<li>使用Alexa Fluor二抗检测一抗（Alexa Fluor 488山羊抗兔，A11008，1：500）。</li>
	<li>使用DAPI抗褪色溶液复染细胞核。</li>
	<li>使用荧光显微镜使用4倍和10倍物镜捕获染色切片的图像和相位图像（见 材料表）。</li>
	<li>对于苏木精和曙红（H&E）染色，在自动染色系统（见 材料表）中处理切片，使用100%，二甲苯95%乙醇，50%乙醇溶液和H&E染色进行脱蜡。</li>
	<li>使用带有明场光源的显微镜使用4倍和10倍物镜拍摄图像。</li>
</ol>

手稿的作者没有潜在的利益冲突需要披露。

人类死后组织是研究人类神经退行性疾病的特别有价值的资源，因为在动物模型中开发的潜在药物的鉴定需要可转化为人类47。人类眼压升高的影响已经得到充分证实，但对异常 ONH 跨层压力变化的研究很少。尽管存在多种动物模型和人类ONH的有限建模，但没有离体人体模型来研究跨层压力变化41，54，55，...

最近调查的全球估计表明，有2.85亿人患有视力障碍，其中包括3900万盲人1。2010 年，世界卫生组织记录发现，列出的九个主要失明原因中有三个发生在眼睛后段1。眼后段疾病累及视网膜、脉络膜和视神经2。视网膜和视神经是大脑的中枢神经系统（CNS）延伸。视网膜神经节细胞（RGC）轴突容易受到损伤，因为它们通过视神经头（ONH）离开眼睛形成视神经3。ONH仍然是RGC轴突最脆弱的点，因为结缔组织束的3D网格作用称为层状（LC）4。ONH是青光眼中RGC轴突损伤的初始部位5，6，7，并且ONH内的基因表达变化已经在眼部高血压和青光眼模型中进行了研究8，9，10。由于眼内隔室（称为眼内压 （IOP））和外眼周蛛网膜腔内（称为颅内压 （ICP））之间的压差，RGC 轴突在 ONH 处易感11。LC区域将两个区域分开，保持正常的压差，IOP范围为10-21 mmHg，ICP范围为5-15 mmHg12。通过两个腔室之间层隙的压力差称为跨层压力梯度（TLPG）13。青光眼的一个主要危险因素是IOP14升高。
增加的IOP会增加层流区域内和层流区域的应变6，15，16。在人类和动物模型中的实验观察表明，ONH是轴突损伤的初始部位17，18。在ONH引起青光眼损伤的IOP相关应力和应变的生物力学范式也影响青光眼的病理生理学19，20，21。尽管在人类中，压力引起的变化会机械地损害RGC轴突22，但叶片内缺乏胶原板的啮齿动物也可能发展为青光眼7，23。此外，IOP 升高仍然是原发性开角型青光眼患者最突出的危险因素，而正常张力型青光眼患者即使没有 IOP 升高也会发生青光眼性视神经病变。此外，还有一部分眼部高血压患者没有视神经损伤。也有人提出，脑脊液压力（CSFp）可能在青光眼发病机制中发挥作用。有证据表明，与正常个体相比，青光眼患者的ICP降低至~5 mmHg，从而导致经层压升高，并在疾病中起关键作用24，25。以前，在犬类模型中已经证明，通过控制IOP和CSFp的变化，视盘可以有很大的位移26。猪眼中CSFp的升高也显示出LC区域和层后神经组织内的主要应变增加。RGC和LC区域的应变增加导致轴突运输阻塞和RGCs的损失27。RGC 的进行性变性与营养支持的丧失28，29、炎症过程/免疫调节刺激30、31 和凋亡效应物有关29、32、33、34、35。此外，轴突损伤（图3）对RGC造成不利影响，引发再生失败36，37，38，39。尽管IOP的影响已经得到了很好的研究，但对异常的跨层压力变化进行了很少的研究。青光眼的大多数治疗方法都集中在稳定眼压上。然而，即使降低眼压可以减缓疾病的进展，它也不能逆转视野丧失并防止RGC的完全丧失。了解青光眼中与压力相关的神经退行性变化对于预防RGC死亡至关重要。
目前的证据表明，由于患有创伤性或神经退行性视力障碍的患者的各种机械，生物学或生理变化引起的跨层压力调节可导致显着的视力丧失。目前，没有真正的临床前人类后段模型可以研究离体人ONH内的青光眼生物力学损伤。观察和治疗眼睛的后段是眼科的巨大挑战27。靶向后眼存在物理和生物屏障，包括高消除率、血视网膜屏障和潜在的免疫反应40。新药靶标的大多数疗效和安全性测试都是利用体外细胞和体内动物模型41完成的。眼部解剖结构很复杂，体外研究不能准确地模拟组织模型系统呈现的解剖学和生理障碍。尽管动物模型是药代动力学研究的必要条件，但人类后眼的眼部生理学可能因各种动物物种而异，包括视网膜的细胞解剖结构，脉管系统和ONH41，42。
使用活体动物需要密集而详细的道德法规、高度的财务承诺和有效的可重复性43。最近，在实验研究中对动物的道德使用提出了其他多项指南44，45，46。动物试验的替代方案是使用离体人眼模型来研究疾病发病机制和保护ONH损伤的药物的潜在分析。人类死后组织是研究人类疾病范式的宝贵资源，特别是在人类神经退行性疾病的情况下，因为在动物模型中开发的潜在药物的鉴定需要可转化为人类47。离体人供体组织已被广泛用于人类疾病的研究47、48、49，人类前段灌注器官培养系统此前为研究IOP升高的病理生理学提供了独特的离体模型50、51、52。
为了研究人眼中与IOP和ICP相关的跨层压，我们成功设计并开发了一种双腔跨层自主系统（TAS），该系统可以使用来自人类供体眼睛的后段独立调节IOP和ICP。它是第一个研究跨层压力并利用TLPG对ONH的生物力学效应的离体人体模型。
这种离体人TAS模型可用于发现和分类由于IOP或ICP慢性升高而发生的细胞和功能修饰。在本报告中，我们详细介绍了剖析，设置和监测TAS人类后段模型的分步方案。该协议将允许其他研究人员有效地复制这种新颖的离体加压人类后段模型，以研究生物力学疾病的发病机制。

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			<td>#122, 1-1/8&#34; Inside x 1-5/16&#34; Outside Diam, Viton O-Ring, 3/32&#34; Thick, 
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			<td>Amazon</td>
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			<td>114 Buna-N O-Ring, 70A Durometer, Black, 5/8&#34; ID, 13/16&#34; OD, 3/32&#34; Width (Pack of 100)</td>
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			<td>B000FMYRHK</td>
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			<td>30 mL Syringes without Needle</td>
			<td>Vitality Medical</td>
			<td>302832</td>
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			<td>3-Way Stopcock, 2 Female Luer Locks, Swivel Male Luer Lock, Vented Cap</td>
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			<td>Corning&#160;L-glutamine Solution</td>
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			<td>Covidien 3033 Curity Gauze Sponge, 4&#34; x 4&#34;, 12-Ply, Sterile, 1200/CS</td>
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			<td>Dumont #5 - Fine Forceps</td>
			<td>F.S.T.</td>
			<td>11254-20</td>
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			<td>Eye Scissors Standard Curved</td>
			<td>Katena</td>
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			<td>Falcon 150 x 15mm Plain Sterile Disposable Petri Dishes</td>
			<td>Capitol Scientific</td>
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			<td>Fisherbrand 4 oz. Specimen Containers</td>
			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
			<td>01-812-54</td>
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			<td>Fisherbrand Instant Sealing Sterilization Pouches</td>
			<td>Fisher Scientific</td>
			<td>01-812-55</td>
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			<td>Fisherbrand Instant Sealing Sterilization Pouches</td>
			<td>Fisher Scientific</td>
			<td>01-812-58</td>
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			<td>HyClone Dulbecco&#39;s Modified Eagles Medium</td>
			<td>Fisher Scientific</td>
			<td>SH3024302</td>
			<td></td>
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			<td>HyClone Penicillin Streptomycin 100X Solution</td>
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			<td>SV30010</td>
			<td></td>
		</tr>
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			<td>Hydrophilic Filter with Female Luer Lock Inlet, Male Luer Slip Outlet, Blue and Clear</td>
			<td>Qosina</td>
			<td>28217</td>
			<td></td>
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			<td>Hydrostatic pressure transducers, DELTRAN &#174; II, Catalog # DPT-200 with a 3CC/HR flow rate</td>
			<td>AD instruments</td>
			<td>DPT-200</td>
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			<td>JG15-0.5HPX 15 Gauge 0.5&#34; NT Premium Series Dispensing Tip 50/Box</td>
			<td>Jenson Global</td>
			<td>JG15-0.5HPX 15</td>
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			<td>Keyence B2&#8208;X710 microscope</td>
			<td>Keyence</td>
			<td>B2-X710</td>
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			<td>LabChart 8</td>
			<td>AD instruments</td>
			<td>LabChart 8</td>
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		<tr>
			<td>Leica ST5020 Multi-stainer</td>
			<td>Leica</td>
			<td>ST5020</td>
			<td></td>
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		<tr>
			<td>Non-Vented Universal Luer Lock Cap, White</td>
			<td>QOSINA</td>
			<td>65811</td>
			<td></td>
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		<tr>
			<td>Octal Bridge Amp (Model # FE228)</td>
			<td>AD instruments</td>
			<td>FE228</td>
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			<td>Pharmco Products ETHYL ALCOHOL, 200 PROOF</td>
			<td>Fisher Scientific</td>
			<td>NC1675398</td>
			<td></td>
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		<tr>
			<td>Phosphate Buffered Solution (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
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		<tr>
			<td>PowerLab 8/35 (Model # PL3508)</td>
			<td>AD instruments</td>
			<td>PL3508</td>
			<td></td>
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			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>ThermoFisher</td>
			<td>P36935</td>
			<td></td>
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			<td>Push-to-Connect Tube Fitting for Air and Water&#160;Straight Adapter, 1/8&#34; Tube OD x 1/8 NPT Male</td>
			<td>McMAster-Carr</td>
			<td>7880T113</td>
			<td></td>
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			<td>Push-to-Connect Tube Fitting with Universal Thread&#160;for Air and Water, Adapter, 1/8&#34; Tube OD x 1/8 Pipe</td>
			<td>McMAster-Carr</td>
			<td>51235K101</td>
			<td></td>
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			<td>Saint-Gobain Tygon S3 E-3603 Flexible Tubing 500 ft.</td>
			<td>Fisher Scientific</td>
			<td>14-171-268</td>
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			<td>Superblock T20</td>
			<td>Fisher Scientific</td>
			<td>PI37536</td>
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			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>F.S.T.</td>
			<td>14001-14</td>
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			<td>Tissue Forceps Delicate 1x2 Teeth Curved</td>
			<td>Katena</td>
			<td>K5-4110</td>
			<td></td>
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			<td>Translaminar Autonomous System (TAS)</td>
			<td>University of North Texas Health Science Center</td>
			<td>N/A</td>
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			<td>USA Size 030 O-ring Buna-N, B1000, 70 Durometer, Black, Buna-N 
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			<td>Marco Rubber &#38; Plastics</td>
			<td>B1000-030</td>
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translaminar autonomous system model for the modulation of intraocular and intracranial pressure in human donor posterior segments

目前对一种新的临床前人类模型的需求尚未得到满足，该模型可以使用颅内压（ICP）和眼内压（IOP）靶向离体疾病病因，该模型可以识别与青光眼发病机制相关的各种致病范式。离体人前段灌注器官培养模型先前已被成功利用并作为青光眼发病机制发现和治疗测试的有效技术。在离体人体器官系统上进行的临床前药物筛选和研究可以更好地转化为临床研究。本文详细介绍了一种称为跨层自主系统（TAS）的新型离体人跨层压力模型的生成和操作。TAS模型可以使用人类供体后段独立调节ICP和IOP。该模型允许以临床前方式研究发病机制。它可以减少活体动物在眼科研究中的使用。与体外实验模型相比，视神经头（ONH）组织结构，复杂性和完整性也可以在离体TAS模型中保持。

跨层自主系统的设计和创建 经层压差是包括青光眼在内的各种疾病发病机制的潜在关键机制。所描述的模型的用途包括但不限于青光眼（眼压升高，可能降低 ICP）、创伤性脑损伤（ICP 升高）和长期暴露于微重力相关视力障碍（ICP 升高、IOP 升高）的研究。为了帮助发现靶向人眼中跨层压的分子发病机制，我们设计、创建并验证了 TAS 模型。我们的新型离体人体模型为独立研究ICP和I...

Watch this Scientific Journal Video about Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments at JoVE.com

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

我们描述并详细介绍了跨层自治系统的使用。该系统利用人后段独立调节节段（眼内）内和视神经周围（颅内）的压力，以产生模仿青光眼性视神经病变特征的跨层压力梯度。

用于调节人类供体后段眼内压和颅内压的跨层自主系统模型

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular ...

translaminar-autonomous-system-model-for-modulation-intraocular

Research

JoVE Journal

Neuroscience

2.9K 次观看.  University of North Texas Health Science Center. 我们描述并详细介绍了跨层自治系统的使用。该系统利用人后段独立调节节段（眼内）内和视神经周围（颅内）的压力，以产生模仿青光眼性视神经病变特征的跨层压力梯度。

视频: Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

科研

神经科学

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

Enrichment of Bruch&#039;s Membrane from Human Donor Eyes

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

从人类供体眼富集布鲁赫的膜

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

自主和可充电 Microneurostimulator 内窥镜植入黏膜下层

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

利用 SPM 软件分析颅内脑电图数据的神经活动和连接性

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...

How to Obtain Reliable Visual Event-related Potentials in Newborns

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable measurements in newborns. Obtaining high-quality, reliable VEPs is crucial for the early detection of abnormal development of the central nervous system in at-risk newborns, and for implementing successful early interventions. Recommendations are based on a previous study which showed that when post-conceptional age, polysomnography-identified sleep stages, and light-emitting diodes (LEDs) googles as the luminous source are controlled, no more than 4 repetitions of VEP averages are required to obtain replicable recordings, variability decreases, and reliable VEPs can be obtained. By controlling for these sources of variability and using statistical analyses, we were able to clearly and reliably identify the amplitude and latency of three main components (NII, PII and NIII) present in 100% of newborns (n = 20) during active sleep. Recording VEPs during awake states, quiet sleep and transitional sleep is not recommended because VEP morphology may differ significantly from one average to the next, leading to the risk of misleading clinical prognoses. Moreover, it is easier to obtain VEPs during active sleep because this state can be clearly and reliably identified at this stage of development, sleep cycles are short enough to allow measurements to be taken in a reasonable time, and the method does not require new o expensive equipment.

Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.

如何获得新生儿中可靠的视觉事件相关潜力

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.

虚拟现实曝光期间颅外直接电流刺激的同步应用

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

斑马鱼游泳期间后横向线阿弗伦特神经元的活动

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Intracranial Pressure Monitoring In Nontraumatic Intraventricular Hemorrhage Rodent Model

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage animal models is essential. In this study, we sought out ways to measure intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during nontraumatic intraventricular hemorrhage in rats. The experimental design included three Sprague Dawley groups: sham, standard 200 &#181;l intraventricular hemorrhage, and vehicle control groups. By introducing an intraparenchymal fiberoptic pressure sensor, precise intracranial pressure measurements were obtained in all groups. Cerebral perfusion pressures were calculated with the knowledge of intracranial pressure and mean arterial pressure values. As expected, the intraventricular hemorrhage and vehicle control groups both experienced a rise in the intracranial pressure and subsequent decline in cerebral perfusion pressure during intraventricular injection of autologous blood and artificial cerebrospinal fluid, respectively. The addition of an intraparenchymal fiberoptic pressure sensor is beneficial in monitoring precise intracranial pressure changes.

Monitoring intracranial pressure in rodent&#160;models of nontraumatic intraventricular hemorrhage is not common in the current literature. Herein, we demonstrate a technique for measuring intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during intraventricular hemorrhage in a rat&#160;animal model.

非创伤性脑室内出血啮齿动物模型中的颅内压监测

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage ...