这项工作得到了国家失聪和其他传播障碍研究所的资助R01DC015824（KMS）和T32DC00038（支持JES和SD），国防部授予W81XWH-14-1-0091（KMS），贝塔雷利基金会（KMS） ，Nancy Sayles Day基金会（KMS），Lauer耳鸣研究中心（KMS）和Barnes Foundation（KMS）。

在手术前获得了收集所有样品的书面知情同意书，根据世界医学协会道德守则（赫尔辛基宣言）进行了实验。研究方案的所有部分均由马萨诸塞州眼科和马萨诸塞州总医院机构审查委员会批准。 注意:以下1-7节设计为在从手术室接收到初级人类VS或GAN样品后依次执行。处理应始终从第1节开始。然后，通常应首先保留RNA（第2部分），然后制备用于收集分泌物（第3部分）和蛋白质提取的组织（第4部分）。要培养的组织（VS的第5部分，GAN的第6部分）可以在补充的培养基中进行，而执行第2,3和4部分。切片组织的固定（第7节）非常短，c在任何离心步骤中进行。第8节（外淋巴处理）和第9节（CSF处理）可以在椎间盘VS切除术期间从手术室接收到外淋巴或CSF后进行。可以在培养中生长的VS或施旺细胞上进行第10节（病毒转导）。 1.准备和接收外科手术样本注意:以下步骤应在无菌环境中进行，例如组织培养罩或层流罩。预计熟悉基本的无菌技术。 <ol><li> 准备用于初级人VS或施万细胞培养的培养基 <ol><li>在37℃水浴中解冻冷冻胎牛血清（FBS）的50mL等分试样。 </li><li>将5mL青霉素/链霉素混合物加入到500mL DMEM / F-12瓶中，最终稀释为1:100。 </li><li>将50mL解冻的FBS加入500mL DMEM / F-12瓶中，最终稀释1:10。 </li><li>在瓶子上写下日期，研究人员的姓名，以及补充信息（ 例如 1％P / S，10％FBS）。 </li><li>将新培养基置于冰箱中（4℃）。 </li></ol></li><li> 接收和清洗VS或GAN样品 <ol><li>在手术室中，将新鲜收获的VS或GAN样品置于标本容器中的无菌盐水中。将样品从手术室的冰上运送到实验室的层流罩。 注意:根据相关肿瘤的大小或切除的神经的数量，患者之间的样本大小和体重差异很大。 </li><li>从冷冻箱中取出透明质酸酶（25,000 U / mL）和胶原酶（3,200 U / mL）的储备溶液等分试样，并使酶在室温下解冻（步骤5.1.4或6.1.6要求，取决于样品是否为一个VS或一个GAN）。 注意:冻干酶的再悬浮在产品信息表中详细描述。可将胶原酶重悬于0.02M磷酸盐缓冲液（pH7.0），77mM NaCl和0.01％1mg / mL牛血清白蛋白溶液中的任何无钙平衡盐溶液和透明质酸酶中。每个冻干酶的比活性随批次而变化，应在再悬浮前进行验证。 </li><li>使用一系列装有1.4mL 1x无菌PBS的1.5mL管，洗涤VS或GAN标本三次。用无菌镊子小心地将样品从管转移到管上。关闭每个管，一旦它包含样品，轻轻摇动洗涤。 注意:为了避免淹没1.5 mL管，如果肿瘤片大，每管可以使用少于1.4 mL的PBS。 </li><li>将样品放入干燥的60 mm培养皿中，并使用镊子去除所有死组织（ 即烧灼部分，通常为白色或不透明的颜色）和具有突出血管的区域。 </li><li>使用f手段或手术刀将样品分成单独的片段，用于RNA保存，蛋白质提取，分泌物收集，固定和细胞培养（见下文第2-6节）。 </li><li>将指定用于细胞培养的试样（第5/6节）转移到含有5-8mL冷的补充DMEM / F-12培养基的新的60mm培养皿中（或者，第一培养皿的盖可以是用于此步骤）。 注意:所需的DMEM / F-12培养基的量（来自步骤1.1）将取决于肿瘤或神经的大小，但应在5至8 mL之间。该片可以在培养基中进行，同时执行后续步骤。 </li></ol></li></ol> 2. RNA保存VS和GAN组织（也适用于人类感觉上皮） <ol><li>在层流罩下，将1-1.2 mL RNA稳定溶液转移到新的1.5 mL管中。 </li><li>用PBS清洗样品（步骤1.2.3）后，短暂地浸泡小馅饼ce的RNA保存（约3毫米3的VS或长度为5毫米的GAN）进入RNA稳定溶液。确保样品完全浸入溶液中。 </li><li>准备并标记一个新的1.5 mL管。从RNA稳定溶液中检索样品，将其转移到新管中，并将其储存在-80°C冷冻箱中。 </li></ol> 3.收集VS和GAN分泌物<ol><li> 第1天 <ol><li>用PBS清洗样品（步骤1.2.3）后，将指定用于收集分泌物的VS或GAN放入空的1.5 mL管中。 </li><li>准备两个额外的1.5 mL管，并将500μL的DMEM（未补充FBS）移液到每个管中。 注意:DMEM的第一管将用于收集肿瘤分泌物，DMEM的第二管将作为匹配对照。分泌物可以收集在DMEM，任何其他典型的培养基中补充血清或​​PBS。 </li><li>将一个含DMEM的管放置在校准的数字刻度上。去皮垢，使得当管放在秤上方时，它读数为0毫克。 </li><li>从秤中取出DMEM管，将指定收集分泌物的试样放入管中，称重。注意结果，这将代表单独的组织的重量。 </li><li>在层流罩下，将管中的DMEM的体积调节为每10毫克样品100μL。 </li><li>将含有组织样品的管和其匹配的对照（单独的DMEM）放入培养箱（37℃，5％CO 2 ）中，与本实验的计划一致。孵育组织至少72小时以收集生物有用量的肿瘤或神经分泌物15 。 </li></ol></li><li> 第4天 <ol><li>孵育72小时后，从中除去含分泌物的培养基标本（ 即组织条件培养基），使用1毫升移液管。为了防止重复的冻融循环，根据计划的下游分析将培养基等分成新的管（ 例如，进行具有4个孔的ELISA的ELISA，每个孔需要50μL，可以制备210μL的等分试样）。 </li><li>如果需要，在-80°C下将原始管中剩余的组织片（已经在第1天标记）冻结，以便进一步分析，如DNA提取。 </li><li>将组织，分泌物和控制DMEM储存在-80°C冷冻箱中，直到下游应用启动（ 例如 ELISA，LC-MS / MS，细胞因子阵列等 ）。 </li></ol></li></ol> 4.从VS或GAN组织提取蛋白质<ol><li> 强化RIPA缓冲液的制备 <ol><li>在一个15 mL的管中，将一片磷酸酶抑制剂和一片蛋白酶抑制剂加入到10mL的RIPA缓冲液中;这是强化的RIPA缓冲区。 注意:将RIPA缓冲区存储在4°C，然后在使用前添加平板电脑。 </li></ol></li><li> 蛋白质提取 <ol><li>将210μL强化的RIPA缓冲液（步骤4.1.1）转移到新的1.5 mL管中。 </li><li>用PBS洗涤样品（步骤1.2.3）后，将指定用于蛋白质提取的小块组织（约3 mm 3的VS或5 mm长度的GAN）转移到含RIPA缓冲液的管中，并置于冰上至少10分钟。如果研究磷酸化形式的蛋白质，补充RIPA缓冲液与蛋白酶和磷酸酶抑制剂14 。同时，可以进行组织处理的其他部件的步骤，例如固定（部分7）。 </li><li>将离心机预冷却至4°C。 </li><li>使用塑料杵，均匀化含有RIPA缓冲液的管中的组织。以20％振幅超声波处理组织10-15秒。确保标本仍然在冰上，即使在超声处理。 </li><li>在15,000 xg和4℃下离心10分钟。 </li><li>将上清液以50μL的增量等分到新的0.5mL管中（根据预期的下游应用，可以选择任何增量大小）。 </li><li>将等分的蛋白质裂解液储存在-80°C冷冻箱中，直到下游应用启动（ 例如， ELISA或Western印迹） 20,21 。 </li></ol></li></ol> 5.初级人类VS文化<ol><li> 第1天 <ol><li>将指定用于细胞培养的VS组织（其已经坐在培养基中，如步骤1.2.6所述）分成约1mm 3片，每只手中使用一对镊子。 </li><li>用10mL血清移液管吸取包含肿瘤片的培养基，并将所有内容物转移到15 mL锥形管中。 </li><li>离心3分钟1,000 xg和25°C。 </li><li>通过将4.7mL补充的DMEM / F-12培养基与250μL胶原酶（终浓度:160U / mL）和50μL透明质酸酶（终浓度:250U / mL）组合，在新的60mm培养皿中制备崩解培养基， ，最终体积为5mL。 注意:两种酶应储存在-20°C的冰箱中，然后在制备此培养基之前解冻（步骤1.2.2）。 </li><li>离心后，肿瘤片将在锥形管底部形成颗粒。仔细移液并丢弃上清液。 </li><li>加入2mL新鲜的含酶崩解介质（步骤5.1.4）加入肿瘤沉淀。上下移动几次以动员组织并将含肿瘤的培养基转移到60mm培养皿中。 </li><li>将培养皿放入培养箱（37℃，5％CO 2 ）中18小时。 </li></ol></li><li> 第二天 <ol><li>温暖D补充有10％FBS和1％青霉素/链霉素（步骤1.1中制备）的MEM / F-12培养基在37℃水浴中。 </li><li>从培养箱中取出含有VS培养物的培养皿（步骤5.1.7）。使用连接到6 mL注射器的22 G针，吸出含培养液的培养基，并将其转移到新的15 mL锥形管中。 </li><li>在1,000×g和37℃下离心5分钟。 </li><li>同时，使用无菌镊子将所需数量的聚D-赖氨酸和层粘连蛋白包被的玻璃盖玻片放入24孔培养板中。 注意:要培养的孔数取决于样品的大小;可以从1cm 3的肿瘤中培养大约24-32个孔，因此对于大多数较小的肿瘤，规划培养细胞的8-16个孔是合适的。 </li><li>离心后，弃上清（ 即富酶培养基），并将残留的细胞沉淀重新悬浮于新鲜的DMEM / F-12培养基中，预热在步骤5.2.1中。 注意:重悬颗粒的体积由步骤5.2.4中计划的孔数确定，估计每个计划孔1 mL的培养基。 </li><li>使用5 mL血清移液管吸出2 mL重悬细胞培养基（步骤5.2.5）。将1mL含肿瘤细胞的培养基沉积到24孔板的每个制备的孔中。 </li><li>继续抽吸和沉积含有肿瘤细胞的培养基（以2mL的增量抽吸，从其中将1mL沉积到每个计划的孔中），直到肿瘤细胞悬浮液在所有制备的孔之间均等分配。 </li><li>将24孔板置于37℃培养箱中过夜。 </li></ol></li><li> 第3天 <ol><li>如果井下有明显的碎屑，请小心地将每个孔中的培养基更换为新的补充DMEM / F-12培养基，注意不要去除贴壁细胞。如果没有，请将板返回孵化器并更换e在第5天第一次。 </li></ol></li><li> 第5-7天及以后 <ol><li>每3天更换培养基。 注意:初级人类VS和GAN细胞通常维持在培养基中，直到其在14天龄或约14天龄的下游应用中使用。 19世纪后，文化纯度下降。 </li></ol></li></ol> 6.原始人类GAN文化<ol><li> 第1天 <ol><li>在解剖显微镜下，从指定用于GAN培养的组织（其已经坐在培养基中，如步骤1.2.6中所概述的）分离出分子束。 <ol><li>通过使用no来拉动神经束来隔离神经束和纤维鞘的束。 5镊子，同时扣上神仙宝碱。 3镊子</li></ol></li><li>一旦分离物与周围的神圣宝石充分隔离，转移到新的培养皿中，该培养皿含有2毫升新鲜的海绵打褶中等。 </li><li>使用手术刀刀片将束切割成1到2毫米的段。 </li><li>将小分子片和周围培养基移入含有3 mL新鲜补充培养基的15 mL试管中，使15 mL试管中的总体积变为5 mL。 </li><li>在1,000 xg和25℃下离心样品3分钟。 </li><li>同时，通过将4.7mL补充的DMEM / F-12培养基，250μL胶原酶和50μL透明质酸酶组合，在新的60mm培养皿中制备雪旺细胞培养基，终体积为5mL。 注意:将两种酶保存在-20°C;在制备该培养基之前在室温下解冻（步骤1.2.2）。 </li><li>在样品离心后，锥体将凝结成锥形管中的小颗粒。丢弃上清液。 </li><li>加入2mL新鲜的含酶崩解介质（步骤6.1.6）加入神经球。移动和d自己多次动员组织并将其转移到新的60毫米培养皿中。 </li><li>将培养皿放在培养箱中（37℃，5％CO 2 ）20-22小时。 注意:这是比VS样品所需的更长的孵化（步骤5.1.7）。 </li></ol></li><li> 第二天 <ol><li>在37°C水浴中加热补充DMEM / F-12培养基。 </li><li>使用连接到6 mL注射器的22 G针头吸取含细胞培养液的培养基，并将其转移到新的15 mL锥形管中。 </li><li>在1,000×g和37℃下离心5分钟。 </li><li>丢弃上清液并将样品重新悬浮在新的预热的DMEM / F-12培养基中。 </li><li>使用无菌镊子将所需数量的涂覆的盖玻片放入24孔培养板的孔中。 注意:每1厘米神经标本计算培养细胞的两个孔促进强壮的培养生长。 </li><li>向每个培养基中加入1mL培养基在24孔板中很好地指定。 </li><li>将24孔板置于37℃和5％CO 2的培养箱中。 </li></ol></li><li> 第3天 <ol><li>如果孔中有明显的碎屑，请用新的补充DMEM / F-12培养基小心地更换培养基，注意不要去除贴壁细胞。如果没有，请将板返回培养箱，并在第5天首次更换培养基。 </li></ol></li><li> 第5-7天及以后 <ol><li>每3天更换培养基。 </li></ol></li></ol> 7. VS＆GAN组织固定<ol><li>将1-1.2mL的4％多聚甲醛（PFA;小心）吸入新的1.5 mL管中。 </li><li>用PBS清洗肿瘤或神经（步骤1.2.3）后，将一小块组织（约3 mm 3的VS或5 mm长度的GAN）转移到4％的PFA中，并将管置于振荡器上4° C。确保样品完全浸没在t中他的解决方案。 </li><li>在固定至少2小时后，从振荡器中取出管，继续进行进一步处理（ 例如，将其嵌入石蜡或最佳切割温度化合物（OCT）中用于随后的免疫组化或免疫荧光） 22,23,24 。或者，如前所述，组织可以快速冷冻， 25,26 。 </li></ol> 8.翻译期间人类外皮的收集和储存<ol><li>将三个无菌的1.5 mL管置于冰上。 注意:一个管子将被保留作为备件，以防污染。 </li><li>向一个1.5 mL的管中加入约1.2 mL PBS溶液，PBS溶液用0.22μm过滤器过滤两次。 注意:为了在椎间盘切除术中收集外淋巴液，外科医生必须首先确保手术卡尔地区没有骨灰，血液或盐水。为了这个目的，外科医生可以在非优势手中使用Schuknecht 21或24 G吸管。 提取外淋巴的一个典型的地方是外侧半规管（但是，根据外科医生的偏好，也可以访问后半规管或圆窗膜）。外科医生应该用金刚石钻头（同时使用连续抽吸）小心地移除迷路骨，以识别半规管的蓝线（ 即膜状迷宫）。半规管通过连接到1mL结核菌素注射器的长27 G针穿过蓝线。 </li><li>请外科医生轻轻吸出少量的外淋巴，然后将注射器和连接的针头通过手术护士。 注意:通常，收集一滴外淋巴或更少的。如果观察到的体积较大，样品可能被其他f污染LUID。 </li><li>在使用前，直接打开准备好的空的1.5 mL管和管填充PBS。打开它们时，注意不要触摸管盖的内部。 </li><li>从外科护士接收注射器和连接的针头，并将流体完全吹入空管，注意不要用针接触管本身。 </li><li>小心地从注射器上取下针头。不要污染针尖，并继续握住一只手。 注意:已经收集到非常小体积的外淋巴，所以一些流体可能会保留在针的长尖端。 </li><li>另一方面，使用注射器本身（无针）将0.2 mL过滤的PBS溶液从准备的管中吸入注射器的体内。 </li><li>将针重新连接到注射器。将注射器完全排空到含有外淋巴的管中，使用无菌PBS冲洗针尖。 </li><li>关闭含有孔和PBS的管并放置<html>它在冰上。 </li><li>将针处置在锐器容器中。 </li><li>将含有外淋巴的试管尽快放入-80°C冰箱。 </li></ol> 9.人类脑脊液的收集和储存<ol><li>将三个（或更多）无菌的1.5 mL管置于冰上。 注意:一个管将作为备份，以防污染或样品体积大于预期。 </li><li>打开硬脑膜后，请外科医生用3 mL注射器（无针头）收集脑脊液。 注意:为防止污染，脑脊液收集应在硬脑膜开口后立即进行。 </li><li>请外科医生将注射器交给外科护士。 </li><li>从外科护士接收注射器，并将所需体积的CSF完全排出到管中。 </li><li>关闭管子并将其放在冰上。 </li><li>将管子尽快放在-80°C的冷冻箱中。 </li></ol>e"&gt; 10。原代VS细胞的病毒转导实验<ol><li>使用病毒储备浓度作为参考，计算拟议的转导实验所需的所需基因组计数（gc）数量和感染复数（MOI）;可将病毒原液直接稀释至补充的培养基中。 注意:可以使用适合于初级细胞转导的任何病毒载体;见Landegger 等 （2017），详细比较六种不同的腺相关病毒血清型27 。此外，当培养原代人细胞时，细胞计数通常在电镀到盖玻片之前不被确定。初级VS细胞对胰蛋白酶消化和传代不良反应，因此为了可靠的MOI计算，可以使用相差显微镜估计每孔的贴壁细胞数。或者，如果细胞接近汇合，则可以通过使用标准细胞密度值可靠地估计它们的数量一个24孔板（ 例如每孔5×10 4个细胞）。 </li><li>在层流罩下，在15mL锥形实验室管中制备含有所需量病毒的补充培养基，使得每个转导细胞孔准备1mL含病毒培养基。上下移动含病毒的培养基，轻轻搅拌均匀。 </li><li>使用无菌镊子，将含有初级VS细胞的盖玻片（步骤5.2.8）从原始的24孔板转移到新的24孔板。每个盖玻片转移后，加入1 mL含病毒的培养基。每次添加培养基时旋转板，以确保细胞均匀涂覆病毒。 </li><li>在计划实验期间，在37℃和5％CO 2下孵育。 注意:48-120小时的孵化都显示出有希望的转导结果27 。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-21+overexpression+contributes+to+vestibular+schwannoma+cell+proliferation+and+survival.">MicroRNA-21 overexpression contributes to vestibular schwannoma cell proliferation and survival.</a> Otol Neurotol. 31 (9), 1455-1462 (2010).</li><li>Doherty, J. K., Ongkeko, W., Crawley, B., Andalibi, A., Ryan, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB+and+Nrg:+potential+molecular+targets+for+vestibular+schwannoma+pharmacotherapy.">ErbB and Nrg: potential molecular targets for vestibular schwannoma pharmacotherapy.</a> Otol Neurotol. 29 (1), 50-57 (2008).</li><li>Aguiar, P. 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作者没有什么可以披露的。

该手稿描述了VS研究的统一方法框架，概述了下游研究应用中人VS和GAN标本的同时处理。随着VS研究进入精密医学的时代，以能够回答许多研究问题的形式准备相同的样本将能够发现特定于个体患者的分子，细胞，遗传和蛋白质组学的见解。 通过免疫细胞化学，使用S100标记阳性的细胞比与DAPI核染色阳性的细胞比例，随时间深入评估人雪旺细胞和VS培养物的纯度。因此，推荐在?...

前庭神经鞘瘤（VSs）是小脑桥角最常见的肿瘤，诊断为每10万人1例。虽然非转移性，但这些肿瘤严重影响患者的生命质量1,2,3,4,5,6 。受影响的人通常生活在听力损失，耳鸣和听觉丰满的感觉之中。随着肿瘤生长，症状越来越虚弱，导致平衡问题，面部麻痹和其他颅神经功能受损。由于脑干压迫导致的危及生命的并发症也可能发生。 VS的管理选择本质上限于监视静脉肿瘤和立体定向放射治疗或用于生长肿瘤的手术切除因为VSs来自外周感觉神经（ 即前庭神经），因此将VS相关观察与从适当的对照神经衍生的神经，如人类大耳神经（GAN）进行比较很重要。健康的GANs在腮腺切除术或颈部解剖中经常被牺牲，可用作健康雪旺氏细胞生理学的健壮模型。 因为没有FDA批准的用于治疗或预防散发性VS的药物，研究人员必须澄清潜在的分子机制确定治疗目标的疾病。已显示在VS发病机制中发挥作用的蛋白质包括merlin，血管内皮生长因子（VEGF），环氧合酶2（COX-2），核因子κB（NF-κB），肿瘤坏死因子α（TNF-α） ，表皮生长因子受体（EGFR）和相关信号分子10,11,12,13,14,15,16,17 。 最近的进展已经扩大和改进了原始人类前庭神经鞘瘤和健康神经组织的收集，处理，培养和下游调查方案18,19 。然而，大多数现有协议被设计为容纳准备这种组织用于单个下游研究应用（ 即单独的细胞培养）。本文提出了一个统一的方法框架，用于同时处理多个下游应用的单个初级人类VS或GAN样品:细胞类型特异性培养，蛋白质提取，RNA保存，肿瘤分泌物收集和组织固定。这项工作还详细介绍了人工脑脊液（CSF）和外翻淋巴结切除术期间的外科手术收集和处理，因为这些密切相关的组织可能作为VS的生物标志物的重要来源。最后，该方案提出了培养中原代人VS细胞病毒转导的步骤，作为该组织用于基因治疗的新应用。

<table><tbody><tr><td>BioCoat Poly-D-Lysine/Laminin 12 mm #1 German Glass Coverslip</td><td>Corning</td><td>354087</td><td>Or prepare coverslips with Corning Laminin (CB-40232) and Cultrex Poly-L-Lysine (3438-100-01)</td></tr><tr><td>CELLSTAR 15 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>188161</td><td></td></tr><tr><td>CELLSTAR 50 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 60 mm</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum, Sterile-filtered</td><td>Sigma-Aldrich</td><td>C1639</td><td></td></tr><tr><td>Costar 24 Well Clear TC-Treated Multiple Well Plates, Sterile</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate, no glutamine, 500 mL</td><td>Thermo Fisher Scientific</td><td>10313-039</td><td></td></tr><tr><td>DMEM/F-12, 500 mL</td><td>Thermo Fisher Scientific</td><td>11320-033</td><td></td></tr><tr><td>Dumont #3 Forceps, Dumoxel</td><td>Fine Science Tools</td><td>11231-30</td><td>Autoclave prior to use</td></tr><tr><td>Dumont #5 Forceps, Standard tip, Inox</td><td>Fine Science Tools</td><td>11251-20</td><td>Autoclave prior to use</td></tr><tr><td>Fetal Bovine Serum, qualified, USDA-approved regions, 500 mL</td><td>Thermo Fisher Scientific</td><td>10437-028</td><td>Aliquot in 50 mL tubes and store in -20&amp;deg;C freezer</td></tr><tr><td>Hyaluronidase from Bovine Testes, Type I-S, Lyophilized Powder</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>Millex-GP Syringe Filter Unit, 0.22 &amp;micro;m, polyethersulfone, 33 mm, sterile</td><td>EMD Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Paraformaldehyde, Reagent Grade, Crystalline</td><td>Sigma-Aldrich</td><td>P6148</td><td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td></tr><tr><td>PBS, pH 7.4, 500 mL</td><td>Thermo Fisher Scientific</td><td>10010-023</td><td>Autoclave prior to use</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL), 100 mL</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>PhosSTOP Phosphatase Inhibitor Tablets</td><td>Roche</td><td>04906845001</td><td></td></tr><tr><td>Pierce Protease Inhibitor Tablets</td><td>Thermo Fisher Scientific</td><td>88666</td><td></td></tr><tr><td>Pipettes and pipette tips, 5/10/25 mL</td><td>Variable</td><td>Variable</td><td></td></tr><tr><td>Plastic Homogenization Pestle for 1.5/2.0 mL Microtubes</td><td>E&amp;amp;K Scientific</td><td>EK-10539</td><td></td></tr><tr><td>PrecisionGlide Needles, 27 G x 1 1/2 in</td><td>BD</td><td>301629</td><td></td></tr><tr><td>RIPA Buffer</td><td>Boston BioProducts</td><td>BP-115</td><td></td></tr><tr><td>RNAlater (RNA stabilization solution)</td><td>Thermo Fisher Scientific</td><td>AM7021</td><td></td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 mL</td><td>Eppendorf</td><td>022363719</td><td>Autoclave prior to use</td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 mL</td><td>Eppendorf</td><td>022363204</td><td>Autoclave prior to use</td></tr><tr><td>Saline - 0.9% Sodium Chloride Injection, bacteriostatic, 20 mL</td><td>Hospira</td><td>0409-1966-05</td><td></td></tr><tr><td>Scalpel Blades - #15</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>Schuknecht Suction Tube 24 gauge</td><td>Bausch + Lomb</td><td>N1698 42</td><td>Useful for the surgical approach (in addition to common otologic surgical instruments) and &lt;em&gt;e.g.&lt;/em&gt; a blue surgical marker</td></tr><tr><td>Specimen Container, OR sterile, 4OZ</td><td>Medline</td><td>DYND30331H</td><td></td></tr><tr><td>Stemi 2000-C Stereo Microscope</td><td>Zeiss</td><td>000000-1106-133</td><td></td></tr><tr><td>Syringe/Needle Combination, Luer-Lok Tip, 5 mL, 22 G x 1 in.</td><td>BD</td><td>309630</td><td></td></tr><tr><td>Tuberculin Syringe Only, Slip tip, 1 mL</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Tuberculin Syringe Only, Slip tip, 3 mL</td><td>BD</td><td>309656</td><td></td></tr><tr><td>Ultrasonic homogenizer, 4710 Series, CV18 probe</td><td>Cole-Parmer</td><td>CP25013</td><td></td></tr></tbody></table>

a unified methodological framework for vestibular schwannoma research

前庭神经鞘瘤是小脑桥角最常见的肿瘤，占所有颅内生长的6-8％。尽管这些肿瘤在高达95％的受影响个体中引起感觉神经性听力损失，但这种听力损失的分子机制仍然难以捉摸。本文概述了我们实验室中建立的步骤，以便于收集和处理各种原始人体组织样品用于前庭神经鞘瘤研究的下游研究应用。具体来说，这项工作描述了从手术样本收集，处理和培养Schwann和神经鞘细胞的统一方法框架。这与现在被认为对当前研究至关重要的并行处理步骤相结合:肿瘤和神经分泌物的收集，RNA的保存和从收集的组织中提取蛋白质，用于制备切片的组织固定，d将原代人细胞暴露于腺相关病毒以用于基因治疗。此外，这项工作突出显示了将这种肿瘤收集在一起的独立手术方法，作为从内耳和外淋巴获得人类感觉上皮的独特机会。提供提高实验质量的提示，并突出显示常见的陷阱。

如第5节所述，培养物中的初级人VS细胞可以被视为许多下游研究应用中疾病相关过程的信息模型（ 图1 ）。在第6节中培养的健康雪旺烷细胞提供了直接和有启发性的比较。如下所述，根据这种统一的方法框架处理的VSs和GAN的广泛数据可以在以前发表的12,13,14,15,27中的多篇文章中找到。 ?...

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A Unified Methodological Framework for Vestibular Schwannoma Research

该方案的目标是概述人类外科手术样品在前庭神经鞘瘤和雪旺氏细胞研究中的多个下游应用的收集和处理。

前庭神经鞘瘤研究的统一方法框架

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these ...

a-unified-methodological-framework-for-vestibular-schwannoma-research

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

7.3K 次观看.  Massachusetts Eye and Ear. 该方案的目标是概述人类外科手术样品在前庭神经鞘瘤和雪旺氏细胞研究中的多个下游应用的收集和处理。 

视频: A Unified Methodological Framework for Vestibular Schwannoma Research

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

三维前庭眼反射测试使用六度自由度运动平台

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

科研

神经科学

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Using Unidirectional Rotations to Improve Vestibular System Asymmetry in Patients with Vestibular Dysfunction

The vestibular system provides information about head movement and mediates reflexes that contribute to balance control and gaze stabilization during daily activities. Vestibular sensors are located in the inner ear on both sides of the head and project to the vestibular nuclei in the brainstem. Vestibular dysfunction is often due to an asymmetry between input from the two sides. This results in asymmetrical neural inputs from the two ears, which can produce an illusion of rotation, manifested as vertigo. The vestibular system has an impressive capacity for compensation, which serves to rebalance how asymmetrical information from the sensory end organs on both sides is processed at the central level. To promote compensation, various rehabilitation programs are used in the clinic; however, they primarily use exercises that improve multisensory integration. Recently, visual-vestibular training has also been used to improve the vestibulo-ocular reflex (VOR) in animals with compensated unilateral lesions. Here, a new method is introduced for rebalancing the vestibular activity on both sides in human subjects. This method consists of five unidirectional rotations in the dark (peak velocity of 320&#176;/s) toward the weaker side. The efficacy of this method was shown in a sequential, double-blinded clinical trial in 16 patients with VOR asymmetry (measured by the directional preponderance in response to sinusoidal rotations). In most cases, VOR asymmetry decreased after a single session, reached normal values within the first two sessions in one week, and the effects lasted up to 6 weeks. The rebalancing effect is due to both an increase in VOR response from the weaker side and a decrease in response from the stronger side. The findings suggest that unidirectional rotation can be used as a supervised rehabilitation method to reduce VOR asymmetry in patients with longstanding vestibular dysfunction.

A new rehabilitation method is presented for rebalancing the vestibular system in patients with asymmetric responses, which consists of unidirectional rotations toward the weaker side. By directly modifying the vestibular pathway rather than enhancing the multisensory aspects of compensation, asymmetry can be normalized within 1-2 sessions and show lasting effects.

使用单向旋转改善前庭功能障碍患者的前庭系统不对称性

The vestibular system provides information about head movement and mediates reflexes that contribute to balance control and gaze stabilization during ...

行为学

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

医学

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

体内Menière 病耳和正常听觉的磁共振成像对人颅神经的形态学分析

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed to be due to the stochastic resonance (SR) phenomenon, which is defined as application of a low-level/subthreshold stimulus to a non-linear system to increase detection of weaker signals. However, it is still unknown how SR exhibits its positive effects on human balance. This is one of the first demonstrations of the effects of sinusoidal and stochastic noise on individual neurons. Using whole-cell patch clamp electrophysiology, sinusoidal and stochastic noise can be applied directly to individual neurons in the medial vestibular nucleus (MVN) of C57BL/6 mice. Here we demonstrate how to determine the threshold of MVN neurons in order to ensure the sinusoidal and stochastic stimuli are subthreshold and from this, determine the effects that each type of noise has on MVN neuronal gain. We show that subthreshold sinusoidal and stochastic noise can modulate the sensitivity of individual neurons in the MVN without affecting basal firing rates.

Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

随机噪声在Vitro内评估中庭前核神经元敏感性的随机噪声应用

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed ...

An Endoscopic Transcanal Transpromontorial Approach for Vestibular Schwannomas using a Computer-Based Three-Dimensional Imaging System

A 2D monocular endoscope has been used in transcanal transpromontory vestibular schwannoma surgery instead of craniotomy. However, the absence of depth perception is the limitation of this approach. With the loss of depth perception, the surgeon will be not able to perform delicate and particularly complicated surgery. A binocular endoscope has been developed to provide stereoscopic vision with better depth perception for complicated anatomic structures and has been applied in some endoscopic surgeries. However, the diameter of the endoscope is a limitation in the performance of transcanal otologic surgeries. A small diameter endoscope facilitates easier surgery in a restricted space. A computer-based 3D imaging system can obtain 3D images in real-time using a small monocular endoscope. In this study, to evaluate the feasibility of a computer-based 3D imaging system for endoscopic lateral skull base surgery, we applied this 3D imaging system in a transcanal transpromontorial approach in two patients with vestibular schwannomas. The surgical procedure was completed without complication in these two cases. There was no mortality, perioperative complications, nor notable postoperative complications. Using this computer-based 3D imaging system, a better depth perception and stereoscopic vision was observed compared to a conventional 2D endoscope. The improvement in depth perception offers superior management of the complicated surgical anatomy.

Here, we present a transcanal transpromontorial approach for vestibular schwannomas using a computer-based three-dimensional (3D) imaging system combined with a two-dimensional (2D) endoscope. This system provided stereoscopic vision, better depth perception and reduced visual fatigue. This 3D imaging system enabled the application of 3D vision technology in endoscopic lateral skull base surgery.

A 2D monocular endoscope has been used in transcanal transpromontory vestibular schwannoma surgery instead of craniotomy. However, the absence of depth ...

Assessment of Static Graviceptive Perception in the Roll-Plane using the Subjective Visual Vertical Paradigm

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow the examination of all semicircular canals in the clinical setting. Assessment methods of the otolithic system, which is responsible for the perception of linear acceleration and perception of gravity, are far less in clinical use. There are several experimental approaches for measuring the perception of gravity. The most frequently used method is the determination of the subjective visual vertical. This is usually measured with the head in an upright position. We present here an assessment method for testing otolith function in the roll plane. The subjective visual vertical is measured in the head upright position as well as with head inclination of &#177; 15&#176; and &#177; 30&#176; in the roll plane. This extended functional paradigm is an easy-to-perform clinical test of otolith function and ensures increased information content for the detection of impaired graviceptive perception.

The perception of gravity is commonly determined by the subjective visual vertical in the head upright position. The additional assessment at head tilts of &#177; 15&#176; and &#177; 30&#176; in the roll plane ensures increased information content for the detection of impaired graviceptive perception.

使用主观视觉垂直范式对卷平面中的静态视觉感知的评估

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow ...

Measuring the Influence of Magnetic Vestibular Stimulation on Nystagmus, Self-Motion Perception, and Cognitive Performance in a 7T MRT

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called magnetic vestibular stimulation (MVS). In this article, we present an experimental setup in a 7T MRT scanner (MRI scanner) that allows the investigation of the influence of strong magnetic fields on nystagmus as well as perceptual and cognitive responses. The strength of MVS is manipulated by altering the head positions of the participants. The orientation of the participants&#39; semicircular canals with respect to the static magnetic field is assessed by combining a 3D magnetometer and 3D constructive interference in steady-state (3D-CISS) images. This approach allows to account for intra- and inter-individual differences in participants&#8217; responses to MVS. In the future, MVS can be useful for clinical research, for example, in the investigation of compensatory processes in vestibular disorders. Furthermore, it could foster insights into the interplay between vestibular information and cognitive processes in terms of spatial cognition and the emergence of self-motion percepts under conflicting sensory information. In fMRI studies, MVS can elicit a possible confounding effect, especially in tasks influenced by vestibular information or in studies comparing vestibular patients with healthy controls.

In this article, we describe the experimental setup, material, and procedures to assess reflexive eye movements, self-motion perception, and cognitive tasks under magnetic vestibular stimulation, as well as the anatomical orientation of the vestibular organs, in a 7 Tesla Magnetic resonance tomography (7T-MRT) scanner.

测量磁前庭刺激对7T MRT眼球震颤、自我运动知觉和认知表现的影响

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called ...

Surgical Treatment of an Endolymphatic Sac Tumor

Endolymphatic sac tumors (ELST) are low-grade papillary adenocarcinoma originating from the endolymphatic sac. Usually slow-growing, with local aggressiveness and a low risk of distant metastases, ELST can be sporadic but also frequently associated with von Hippel Lindau disease. The current treatment of ELST is primarily surgical resection. A 55-year-old woman accessed our otologic tertiary level referral center for a sudden worsening of hearing loss in her left ear and vertigo. A magnetic resonance (MRI) and computer tomography scan study subsequently showed a mass in the petrous bone; hence, the presence of an ELST was hypothesized. After embolization of the mass, the patient underwent surgical removal of the lesion. The resection of the mass was done through a translabirinthine approach, with an uneventful procedure. No residual disease remained after surgery. After 24 months of radiologic follow up with MRI, there are no signs of recurrence disease. This paper reports the management of this sporadic ELST, as well as the follow up results, providing clinicians this protocol for the handling of such a challenging otologic skull base surgery and rare disease.

Here, we discuss clinical and radiological features of endolymphatic sac tumors and report the methodology and results of a surgical removal in a case in care at our otologic referral center.