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。

<table><tbody><tr><td>#122, 1-1/8&quot; Inside x 1-5/16&quot; Outside Diam, Viton O-Ring, 3/32&quot; Thick,&lt;br/&gt; 755 Durometer 50 Pack</td><td>Amazon</td><td>B07DRGPPZJ</td><td></td></tr><tr><td>114 Buna-N O-Ring, 70A Durometer, Black, 5/8&quot; ID, 13/16&quot; OD, 3/32&quot; Width (Pack of 100)</td><td>Amazon</td><td>B000FMYRHK</td><td></td></tr><tr><td>30 mL Syringes without Needle</td><td>Vitality Medical</td><td>302832</td><td></td></tr><tr><td>3-Way Stopcock, 2 Female Luer Locks, Swivel Male Luer Lock, Vented Cap</td><td>QOSINA</td><td>2C6201</td><td></td></tr><tr><td>4-40 X 1/2 PH PAN MS SS/CHROME &amp;amp; appropriate sized phillips screwdriver</td><td>Brikksen Stainless Steel Fastners</td><td>PPMSSSCH4C.5</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>ANPROLENE 16 LARGE AMPULE</td><td>Fisher Scientific</td><td>NC9085343</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Betadine</td><td>Purdue</td><td>PUR1815001EACH</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Corning 100 x 20mm tissue-culture treated culture dishes</td><td>Sigma-Aldrich</td><td>CLS430167-100EA</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Corning&amp;nbsp;L-glutamine Solution</td><td>Fisher Scientific</td><td>MT25005CI</td><td></td></tr><tr><td>Covidien 3033 Curity Gauze Sponge, 4&quot; x 4&quot;, 12-Ply, Sterile, 1200/CS</td><td>Med Plus Medical Supply</td><td>COV-3033-CS</td><td></td></tr><tr><td>Dressing Forceps Delicate Curved (serrated)</td><td>Katena</td><td>K5-4010</td><td></td></tr><tr><td>Dumont #5 - Fine Forceps</td><td>F.S.T.</td><td>11254-20</td><td></td></tr><tr><td>Eye Scissors Standard Curved</td><td>Katena</td><td>K4-7410</td><td></td></tr><tr><td>Falcon 150 x 15mm Plain Sterile Disposable Petri Dishes</td><td>Capitol Scientific</td><td>351058</td><td></td></tr><tr><td>Fisherbrand 4 oz. Specimen Containers</td><td>Fisher Scientific</td><td>16-320-730</td><td></td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouches</td><td>Fisher Scientific</td><td>01-812-54</td><td></td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouches</td><td>Fisher Scientific</td><td>01-812-55</td><td></td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouches</td><td>Fisher Scientific</td><td>01-812-58</td><td></td></tr><tr><td>HyClone Dulbecco&#039;s Modified Eagles Medium</td><td>Fisher Scientific</td><td>SH3024302</td><td></td></tr><tr><td>HyClone Penicillin Streptomycin 100X Solution</td><td>Fisher Scientific</td><td>SV30010</td><td></td></tr><tr><td>Hydrophilic Filter with Female Luer Lock Inlet, Male Luer Slip Outlet, Blue and Clear</td><td>Qosina</td><td>28217</td><td></td></tr><tr><td>Hydrostatic pressure transducers, DELTRAN &amp;reg; II, Catalog # DPT-200 with a 3CC/HR flow rate</td><td>AD instruments</td><td>DPT-200</td><td></td></tr><tr><td>JG15-0.5HPX 15 Gauge 0.5&quot; NT Premium Series Dispensing Tip 50/Box</td><td>Jenson Global</td><td>JG15-0.5HPX 15</td><td></td></tr><tr><td>Keyence B2?X710 microscope</td><td>Keyence</td><td>B2-X710</td><td></td></tr><tr><td>LabChart 8</td><td>AD instruments</td><td>LabChart 8</td><td></td></tr><tr><td>Leica ST5020 Multi-stainer</td><td>Leica</td><td>ST5020</td><td></td></tr><tr><td>Non-Vented Universal Luer Lock Cap, White</td><td>QOSINA</td><td>65811</td><td></td></tr><tr><td>Octal Bridge Amp (Model # FE228)</td><td>AD instruments</td><td>FE228</td><td></td></tr><tr><td>Pharmco Products ETHYL ALCOHOL, 200 PROOF</td><td>Fisher Scientific</td><td>NC1675398</td><td></td></tr><tr><td>Phosphate Buffered Solution (PBS)</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td></td></tr><tr><td>PowerLab 8/35 (Model # PL3508)</td><td>AD instruments</td><td>PL3508</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>ThermoFisher</td><td>P36935</td><td></td></tr><tr><td>Push-to-Connect Tube Fitting for Air and Water&amp;nbsp;Straight Adapter, 1/8&quot; Tube OD x 1/8 NPT Male</td><td>McMAster-Carr</td><td>7880T113</td><td></td></tr><tr><td>Push-to-Connect Tube Fitting with Universal Thread&amp;nbsp;for Air and Water, Adapter, 1/8&quot; Tube OD x 1/8 Pipe</td><td>McMAster-Carr</td><td>51235K101</td><td></td></tr><tr><td>Saint-Gobain Tygon S3 E-3603 Flexible Tubing 500 ft.</td><td>Fisher Scientific</td><td>14-171-268</td><td></td></tr><tr><td>Superblock T20</td><td>Fisher Scientific</td><td>PI37536</td><td></td></tr><tr><td>Surgical Scissors - Sharp-Blunt</td><td>F.S.T.</td><td>14001-14</td><td></td></tr><tr><td>Tissue Forceps Delicate 1x2 Teeth Curved</td><td>Katena</td><td>K5-4110</td><td></td></tr><tr><td>Translaminar Autonomous System (TAS)</td><td>University of North Texas Health Science Center</td><td>N/A</td><td></td></tr><tr><td>USA Size 030 O-ring Buna-N, B1000, 70 Durometer, Black, Buna-N&lt;br/&gt; (NBR, Nitrile, Buna)</td><td>Marco Rubber &amp;amp; Plastics</td><td>B1000-030</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.0K 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

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

小梁响应压升高在活的人类眼睛

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

科研

医学

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

跟踪开放全球伤害和治疗性能的前段器官文化平台

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

生物工程

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

小鼠中可逆硅油诱发眼部高血压模型

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

神经科学

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

Source: B&#246;mers, J. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/62567">Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure for establishing a subarachnoid hemorrhage rat model. An anesthetized rat is implanted with probes to measure intracranial pressure and cerebral blood flow. The target site is identified, and the needle is inserted at a pre-chiasmatic position. Autologous blood is then injected into the subarachnoid space, increasing intracranial pressure and decreasing cerebral blood flow, establishing the model.

Source: B&#246;mers, J. et al. Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model. J. Vis. ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

 Induction of Elevated Intraocular Pressure in a Rat Model

<a href='https://appjove.unicartagenaproxy.elogim.com/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)</a>This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison.

Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)This ...