作者感谢达特茅斯比较医学和研究中心（CCMR）的工作人员对用于这些研究的小鼠的专业护理。伯恩斯坦研究基金资助了这项研究。

所有动物工作都采用达特茅斯盖泽尔医学院机构动物护理和使用委员会（IACUC）审查和批准的协议。
1. 处理脑和脊髓样本进行脱钙
<ol><li>分离脑和脊髓样本
	<ol><li>通过吸入CO2 对小鼠实施安乐死。确保 CO2 流速每分钟置换保持架容积的 10%–30%。</li>
		<li>使用镊子提起剑突，并用剪刀在肋骨下方横向切割腹壁，向上拉以避免切割下面的血管或器官。横向切开隔膜。</li>
		<li>沿着平行于肺部的侧缘切开肋骨，直至锁骨。使用镊子抬起胸骨并用止血器夹住胸骨。将止血器放在头上，将胸腔抬开并露出心脏。</li>
		<li>使用镊子抓住心脏的顶点附近，并在心脏的右心房做一个切口以提供出口。将连接 10 mL 注射器的 25 G 针头缓慢施用 10 mL 冰冷的 1x 磷酸盐缓冲盐水 （PBS） 注入左心室以经心灌注小鼠。 注意:灌注应在4-5分钟内进行，直到肝脏清除血液。总共 10 mL 的 1x PBS 通常足以进行灌注，但如有必要，可以使用更多。肝脏透明通常表明灌注充分。</li>
		<li>使用锋利的剪刀，通过斩首去除头部（图1;1）。在皮肤上做一个中线切口（图1;2），然后将皮肤翻转到眼睛上以释放颅骨。</li>
		<li>在鼻骨处切开以从颅骨中释放下颌骨（图1;3）。切除下颌骨、舌头和眼睛。沿着颅骨的侧面切开，沿外耳道释放组织（图1;4）。修剪以去除覆盖颅骨的所有多余皮肤、肌肉和组织。</li>
		<li>用锋利的剪刀平行于脊柱切割，将肋骨与脊柱分开（图1;5和6）。在下腰部区域做一个小切口以隔离脊柱（图1;7）。修剪并去除脊柱上任何剩余的肌肉，以露出椎骨（图1;8）。 注意:从脊柱和颅骨中去除多余的组织对于获得固定剂多聚甲醛（PFA）和乙二胺四乙酸（EDTA）脱钙缓冲液的充分渗透是必要的。</li>
	</ol></li>
	<li>固定后、脱钙和冷冻保存
	<ol><li>使用镊子，将具有完整颅骨或脊柱的大脑放入含有 10 mL 4% PFA 的 15 mL 锥形管中。将试管置于4°C至少48小时以充分固定。 注意:为避免组织过度固定，请勿超过固定72小时。骨标本脱钙前的固定时间延长。充分的固定将保护组织免受脱钙的影响，并确保更好的组织形态。</li>
		<li>用镊子从 4% PFA 中取出组织并将组织放入带有 10 mL 1x PBS 的一次性 14 mL 管中冲洗大脑或脊髓 5 分钟。将脑或脊髓转移到装有 10 mL 10% EDTA 的 50 mL 锥形管中 （pH = 7.2–7.4）。 注意:使用含有 10 mL EDTA 的较大管可使 EDTA 与组织具有更大的接触面积并加速脱钙过程。</li>
		<li>每天检查骨骼是否柔软柔韧:用镊子从EDTA溶液中取出组织，将其放在培养皿上，然后用25 G针轻轻测试骨骼柔软度。如果针头容易穿透骨骼，则脱钙过程完成。</li>
		<li>从EDTA溶液中取出组织，并将脑或脊髓转移到含有10mL 1x PBS的14 mL一次性管中，并洗涤10分钟。重复洗涤。 注意:脱钙通常需要2-3天。如果骨骼尚未充分脱钙，则应每 2-3 天更换一次溶液。然而，在骨脱钙后在EDTA中长时间孵育会损害组织形态。</li>
		<li>通过将蔗糖添加到 1x PBS 中来制备 10%、20% 和 30% 蔗糖溶液。例如，对于 10% 蔗糖，加入 10 g 蔗糖，并使用无菌 1x PBS 将体积增加到 100 mL。将溶液在4°C下储存长达1个月。 注意:蔗糖溶液容易滋生微生物，因此样品不应在这些溶液中长时间储存。</li>
		<li>从1x PBS中取出组织，将其放入10mL的10%蔗糖溶液中，并将其储存在4°C。让组织静置24小时或直到它沉入管底部。</li>
		<li>重复此过程，首先将组织移至20%蔗糖溶液中，最后移至30%蔗糖溶液中。让组织沉入30%蔗糖（至少24小时）并进行组织包埋。</li>
	</ol></li>
	<li>组织包埋和冷冻
	<ol><li>使用镊子，从30%蔗糖中取出组织，将其放在培养皿上，然后倾斜培养皿以去除组织上多余的蔗糖溶液。使用手术刀将组织切成所需的段。</li>
		<li>在冷冻机底部形成一层薄薄的最佳切割温度（OCT）化合物，并将组织片放入模具中。用OCT化合物完全覆盖组织，确保没有气泡。</li>
		<li>通过将悬停在液氮38 上或将块放在100%异丙醇/干冰浆料39 上来快速冷冻块，直到块不透明。将冷冻模具包裹在铝箔中，并将块储存在-80°C下以便长期储存。切片前将块移至-20°C。 注意:在对脱钙的大脑进行切片和执行组织学方案时必须小心，因为如果切片处理不当，颅骨和脑膜层可能会丢失。</li>
	</ol></li>
</ol>2. 脑膜和中枢神经系统组织的制备，用于流式细胞术染色
<ol><li>提取颅骨和大脑
	<ol><li>使用锋利的剪刀，通过斩首去除头部（图2A;1）。使用剪刀在皮肤上做一个中线切口（图2A;2），然后将皮肤翻转到眼睛上以释放头骨。</li>
		<li>将剪刀放在大孔内，开始沿着皮质向嗅球横向切割颅骨，将切口保持在外耳道和下颌骨上方（图2A; 3）。在另一侧进行相同的切割，切口在嗅球处相遇，以将颅骨帽从大脑中释放出来（图2A; 3）。</li>
		<li>使用镊子，剥开颅骨盖骨并将颅骨盖骨放入含有 5 mL 冷 RPMI 培养基和补充有 25 mM HEPES 的 15 mL 锥形管中。将管子放在冰上。</li>
		<li>使用弯曲的镊子，将镊子放在大脑底部下方，然后提起以使大脑从颅帽中解放出来。将大脑放入含有 5 mL 冷 RPMI 和补充有 25 mM HEPES 的 15 mL 锥形管中。将试管放在冰上直到处理。</li>
	</ol></li>
	<li>提取脊柱和脊髓组织
	<ol><li>使用镊子和锋利的剪刀，通过平行于脊柱切割将肋骨与脊柱分开（图2A;4和5）。在下腰部区域做一个小切口以隔离椎柱（图2A; 6）。修剪并去除脊柱上任何剩余的肌肉，以暴露椎骨（图2A;7）。</li>
		<li>将超细手术剪刀放在椎柱内，并沿着柱子的侧边缘切割（图2C）。完全切开对侧边缘，将椎柱分为前部和后部。 注意:脊髓将保持附着在脊柱上。</li>
		<li>使用镊子，缓慢而小心地从椎柱上剥离脊髓，并将组织放入含有 5 mL 冷 RPMI 和 25 mM HEPES 的 15 mL 锥形管中。将脊柱的前部和后部转移到含有 5 mL 冷 RPMI 和 25 mM HEPES 的 15 mL 锥形管中。</li>
	</ol></li>
	<li>去除脑膜以制备单细胞悬液
	<ol><li>使用镊子，从 RPMI 培养基上取下颅骨帽。使用锋利的镊子（#7 镊子; 材料表），在颅骨帽外缘周围划痕（图2B），并将脑膜从颅帽边缘剥离，刮擦以去除硬脑膜和蛛网膜脑膜。将脑膜放在培养皿上。 注意:从大脑和脊髓中去除脑膜需要练习。如果用户在提取脑膜时遇到困难，请使用解剖显微镜来帮助去除。</li>
		<li>从管中取出椎柱。使用锋利的镊子，在椎柱边缘划伤以释放脑膜，并使用弯曲的镊子从椎骨边缘剥离脑膜。将脑膜放在培养皿上。</li>
		<li>将尼龙网状过滤器放入 50 mL 锥形管中。将脑膜移入过滤器中，加入 3 mL 的 RPMI 和补充有 25 mM HEPES。使用 5 mL 注射器的柱塞，通过过滤器研磨组织和培养基。</li>
		<li>使用 5 mL 血清移液器，再用 2 至 3 mL RPMI/HEPES 培养基清洗过滤器，直到所有可见组织都通过过滤器。 注意:为了获得足够的细胞数用于流式细胞术分析，可能需要将来自多只动物的脑膜合并在一起。在这个实验中（图3和图 4），将 4-5只小鼠的大脑和脊髓脑膜汇集在一起。如果样品是混合的，则需要额外的培养基通过尼龙网过滤器研磨组织，以防止组织过热。</li>
		<li>使用 10 mL 血清移液器，将细胞和培养基转移到新鲜的 15 mL 锥形管中。使用 10 mL 血清移液器，用 5 mL 培养基洗涤 50 mL 锥形管以收集任何剩余细胞。在4°C下以450× g 离心5分钟以沉淀细胞。</li>
		<li>使用带真空的巴斯德移液器，吸出上清液，小心避开细胞沉淀，并将细胞重悬于适当的体积和缓冲液中。</li>
		<li>要计数单细胞悬液，请使用台盼蓝排除染料（1:10 稀释）和 RPMI 稀释少量细胞（即 5–10 μL）。向血细胞计数器中加入 10 μL 稀释液。</li>
		<li>如前所述对单元格进行计数40,41，取平均值至少两个 16 平方网格以确保准确性。 注意:例如，在 图3中，将来自脑膜的混合颗粒细胞重悬于250μL荧光激活细胞分选（FACS）缓冲液（1x PBS，含1%FBS）中，用于下游表面染色。将细胞按1:10稀释以进行计数（5 μL细胞，5 μL台盼蓝，40 μL RPMI）。这种稀释每16个方形网格产生50-100个细胞，确保更准确的细胞计数，因为细胞既不太致密和重叠，也不太稀疏。每只小鼠来自汇集脑和脊髓脑膜的有核细胞计数如下:假治疗小鼠的脑膜= 100,000-150,000个细胞和来自Theiler小鼠脑脊髓炎病毒诱导的脱髓鞘疾病（TMEV-IDD）小鼠的脑膜= 300,000-350,000个细胞。细胞计数将根据收集、处理的精度以及是否存在脑膜炎症而有所不同。</li>
		<li>继续进行所需的单细胞技术，例如FACS表面（图3）14,36,42,43,44，细胞内染色方案33,45，体外刺激，细胞培养测定33,46,47，ELISPOT测定28,34,35以及块或单细胞转录组学37,48。 注意:在处理步骤之间将所有试管放在冰上。</li>
	</ol></li>
	<li>制备脑和脊髓组织的单细胞悬液
	<ol><li>通过从管中倒出组织和培养基，将带有培养基的脑或脊髓组织转移到100mm培养皿的顶部。使用镊子将组织移动到培养皿的底部。用无菌剃须刀片将大脑或脊髓切碎。使用剃须刀片，通过刮擦将切碎的组织移动到板的底部以收集组织。 注意:对于下面的酶消化方案，最多可以将两条脊髓汇集在一起进行处理。大脑应该单独处理。</li>
		<li>使用 5 mL 血清移液器，向培养皿中加入 3 mL 补充有 10% 胎牛血清 （FCS） 的 RPMI。使用 10 mL 血清移液器上下移液，将组织重悬于培养基中，并转移到 15 mL 锥形管中。 注意:如果下游应用是单细胞或批量RNA测序分析或细胞培养，则应测试FCS批次以确保在分析前未激活细胞。或者，可以使用含有0.04%BSA的1x PBS而不是含有10%FCS的RPMI来处理细胞。</li>
		<li>使用 10 mL 血清移液器，用另外 2 mL 培养基洗涤培养皿以收集任何残留组织并转移到锥形管中，总体积为 5 mL。在加工步骤之间将试管放在冰上。</li>
		<li>使用移液管将胶原酶I粉末重悬于Hank平衡盐溶液（HBSS）培养基中，以获得所需的浓度（即100mg / mL）。将I型胶原酶添加到含有切碎组织样品的锥形管中，以获得所需的最终浓度（即，50μL，1mg / mL）。 注意:较高浓度的胶原酶将增加细胞产量，但会切割细胞表面标志物。因此，应滴定胶原酶I批次，以确定获得所有必需细胞表面标志物完好无损的情况下获得最大数量活细胞所需的最佳浓度。例如，在单个脑或脊髓样品上以0.5 mg/mL、1 mg/mL和2 mg/mL的终浓度测试胶原酶I。使用台盼蓝排除法测定细胞活力，并通过流式细胞术评估细胞表面标志物CD45、CD19和CD4。1 mg/mL浓度的胶原酶I产生最高的活细胞计数，同时保留所有感兴趣的细胞表面标志物。因此，该浓度用于检查这些细胞类型的进一步实验。</li>
		<li>使用0.15M氯化钠轻轻重悬DNase I粉末至所需的储备浓度。将重悬的 DNase I 加入包含切碎组织样品的锥形管中，以获得 20 U/mL 的终浓度。 注意:脱氧核糖核酸酶I批次因每毫升的活性单位而异。要添加到组织样品中的浓度将根据储备小瓶每毫升的活性单位而变化。每个样品的最终所需浓度为 20 U/mL。</li>
		<li>将试管置于37°C水浴中的管架中并孵育40分钟。每15分钟倒置试管，使组织与酶彻底混合。孵育后，向每个试管中加入 500 μL 0.1 M EDTA （pH = 7.2），终浓度为 0.01 M EDTA，并再孵育 5 分钟以灭活胶原酶。</li>
		<li>使用 10 mL 血清移液器，向每个试管中加入 9 mL 补充有 10% FCS 的 RPMI，使每个试管的体积达到 ~14.5 mL。在4°C下以450× g 离心5分钟。使用真空巴斯德移液管，吸出上清液，小心不要接触细胞沉淀。</li>
		<li>使用 5 mL 血清移液器，向含有细胞沉淀的试管中加入 3 mL 100% 储备等渗密度梯度溶液。使用 10 mL 血清移液器，添加额外的 RPMI 10% FCS 培养基，使最终体积达到 10 mL，并重悬细胞沉淀以创建 30% 储备等渗密度梯度溶液层。 注意:提前准备100%储备等渗密度梯度培养基，等分，并在4°C下储存长达3个月。为了制备100%储备等渗密度梯度溶液，用密度梯度介质稀释缓冲液稀释密度梯度培养基（材料表）。制备密度梯度培养基稀释缓冲液（80.0 g/L NaCl、3.0 g/L KCl;0.73 g/L Na 2 HP0 4、0.20 g/L KH2HP04;20.0 g/L 葡萄糖）并使用真空过滤系统过滤灭菌。通过混合1份密度梯度稀释缓冲液和9份密度梯度介质，制成100%储备等渗密度梯度溶液。混合均匀。</li>
		<li>在添加 70% 原液等渗密度梯度溶液垫层之前，将每个管倒置并充分混合。将含有 1 mL 70% 原液等渗密度梯度溶液的 1 mL 血清移液器插入试管底部。慢慢地在1 mL溶液下垫，注意不要产生气泡。慢慢从管中取出血清移液管，注意不要干扰梯度。 注意:对于 70% 衬垫，应使用 RPMI 培养基将 100% 储备等渗密度梯度溶液稀释至 70%（即，7 mL 100% 储备等渗密度梯度溶液和 3 mL RPMI 培养基充分混合）。此外，创建干净、不受干扰的 70% 衬垫对于去除髓鞘碎片和离心后在梯度界面获得纯单细胞悬浮液至关重要。</li>
		<li>在4°C下以800× g 离心30分钟，没有刹车。吸出上清液，包括髓鞘碎片层，直到 2-3 mL 留在管中，注意不要干扰细胞层。使用 1 mL 移液器在 30/70% 密度梯度之间收获细胞层，并转移到新的 15 mL 锥形管中。</li>
		<li>使用 10 mL 血清移液器，加入 RPMI 10% FCS 培养基，使最终体积达到 15 mL。在4°C下离心450× g5 分钟。 注意:在此步骤中，如果需要，可以汇集来自两个管的细胞层。不要汇集超过两个试管，否则由于高密度梯度培养基浓度，细胞不会沉淀。</li>
		<li>小心吸出上清液，以免干扰细胞沉淀。将细胞重悬于适当的体积/缓冲液中，以使用血细胞计数器对细胞进行计数（例如，将单条脊髓重悬于250μL FACS缓冲液中以进行下游表面染色）（图3）。</li>
		<li>使用 1 mL 移液器，将细胞悬浮液转移到过滤顶部管的顶部（材料表），并让细胞过滤到管底部以去除任何残留的髓磷脂碎片。</li>
		<li>使用台盼蓝排阻染料，通过平均至少两个 16 个方形网格来稀释并计数血细胞计数器上的细胞，准确度为 40,41。</li>
		<li>继续使用所需的单细胞分析技术。 注意:使用各种形式的胶原酶（即D，I型，II型，IV型），免疫细胞，小胶质细胞（图3），星形胶质细胞，周细胞，内皮细胞49和神经元50 都可以有效地分离。使用滴定胶原酶I酶获得的结果的有核细胞计数如下:整个假处理的大脑= 500,000-600,000个细胞;整个TMEV-IDD大脑= 800,000-1,000,000个细胞;整个假处理的脊髓= 150,000-200,000个细胞;整个TMEV-IDD脊髓= 300,000–400,000。细胞计数将根据收集、处理的精度以及是否存在 CNS 炎症而有所不同。</li>
	</ol></li>
</ol>

作者没有什么可透露的。

在稳态和疾病期间评估CNS区室细胞组成的方法对于了解CNS的生理和病理状态至关重要。然而，尽管是中枢神经系统的重要屏障并容纳多种免疫细胞，但脑膜经常被省略在分析中，因为许多传统的大脑和脊髓组织提取方法不允许收集这些膜。这种遗漏是我们了解脑膜细胞组成和功能及其在稳态和炎症条件下的作用的关键限制。最近的研究表明，驻留在脑膜隔室中的常驻和外周来源的免疫细胞在维持中枢神经系统的稳态以及驱动中枢神经系统疾病发病机制方面起着至关重要的作用。这些研究强调不仅需要分析实质隔室，还需要分析周围的脑膜层。这里描述的方案允许在保留脑膜的同时快速分离脑和脊髓，最终能够利用组织学和单细胞研究对CNS区室进行全面的下游分析。代表性结果集中在评估中枢神经系统区室中的免疫细胞;然而，该方案可能适用于分析小胶质细胞、星形胶质细胞、神经元、周细胞、内皮细胞或其他中枢神经系统驻留细胞。
所述方法中的一个关键步骤是仔细提取组织，这对于从脑、脊髓和脑膜中分离纯单细胞悬液以及获得具有完整颅骨或椎柱的中枢神经系统组织至关重要，从而实现高质量的组织形态。实践获得单细胞悬液的技术是成功组织提取的关键，因为它不仅可以提高样品的纯度，还可以提高下游应用的细胞产量。同样，分离具有完整颅骨或椎柱的中枢神经系统组织以充分去除骨骼周围的多余组织是确保更好地固定、脱钙和冷冻保存组织的重要步骤，这些都是获得具有高质量形态和完整抗原靶标的组织切片的关键组成部分。
我们从脑膜中分离单细胞悬浮液的方案的一个局限性是，它侧重于获得硬脑膜和蛛网膜脑膜32,54，同时省略了PIA的分离，PIA通过星形胶质细胞过程仍然附着在大脑或脊髓上。尽管驻留在软脑膜中的细胞是脑膜隔室的重要组成部分，但由于充分分离它需要大量时间，因此决定在单细胞悬液制备期间排除软脑膜55。同样，该方案仅侧重于在颅骨上部获得脑膜，并排除在大脑和脉络丛中分离内陷脑膜。内陷脑膜和脉络丛的收集可以根据先前发布的协议55进行，尽管这是一个耗时的过程。在这两种情况下，选择省略部分脑膜层是由于分离它们需要大量的时间。用于制备下游应用（如流式细胞术、细胞培养测定和 RNA 测序 （RNAseq））所需的单细胞悬液的方案时间对于提高细胞活力和数据质量至关重要。因此，根据具体的研究问题，应在样品分离时间和提取脑膜的彻底性之间取得平衡。在目前的方案中，仅从硬脑膜和蛛网膜脑膜中获得单细胞悬浮制剂，这限制了将结果外推到整个CNS脑膜的能力。然而，如果将这些方法与具有三层脑膜的整个组织切片的IHC或ISH分析结合使用，则可以更全面地了解实质和脑膜隔室中的细胞和分子动力学。
该方案的另一个限制是从硬脑膜和蛛网膜脑膜获得的细胞数量低，特别是在稳态条件下。但是，如果脊髓、大脑或脑膜中的细胞数量被认为太低，无法分析特定的靶细胞群，则可以通过估计给定精度所需的事件总数来确定该分析所需的最小细胞数（例如，5%变异系数），如以前专门从事罕见事件分析的文献中所述56.然后，这些计算可用于确定是否必须汇集来自多只动物的样品，以获得足够的细胞数量来分析感兴趣的细胞群。
所描述的方法通过扩展分析以包括通常被忽视的脑膜隔室，为研究CNS隔间的细胞动力学提供了重要的进展。这些方案允许单独提取大脑、脊髓、硬脑膜和蛛网膜脑膜以产生单细胞悬液。此外，脱钙方案允许对包括三个脑膜层的脑或脊髓组织组成的组织切片进行组织学分析。利用EDTA提供了一个缓慢但温和的脱钙过程，最适合需要高质量组织形态的标本（例如，IHC和ISH）。该方案使用7.2-7.4的pH值来减缓脱钙速率并确保维持完整的组织形态，这是与强酸和弱酸（例如盐酸和三氯乙酸）相比的明显优势，后者具有较短的脱钙时间，但会影响组织形态的质量。
寻求从脑膜中分离单细胞悬浮液的未来方法应侧重于制定方案，以缩短从中枢神经系统组织和大脑中内陷脑膜中彻底分离皮膜层所需的时间。获得三个脑膜层的完整复合物不仅可以增加任何分析的细胞数量，还可以对占据脑膜的常驻和浸润免疫细胞进行更全面的评估，这些免疫细胞包围着中枢神经系统。同时，未来专注于制备脱钙脑和脊髓的方案应寻求确定旨在加速组织脱钙同时保持组织形态质量的新型药物。然而，总的来说，分析CNS单细胞悬液（包括双脑膜和蛛网膜）的下游应用，结合全面的组织切片分析，可以提供对CNS区室内的细胞表型，功能和定位的详细理解。

中枢神经系统（CNS）是一个免疫学专业化的部位。中枢神经系统实质（不包括脑脊液间隙、脑膜和脉管系统）通常被视为免疫特权部位 1,2,3,4,5，在稳态条件下相对缺乏免疫细胞 2,6,7。相比之下，由硬脑膜、蛛网膜和软脑膜层组成的脑膜是中枢神经系统隔室的重要组成部分，在疾病发病机制期间积极参与稳态免疫监视和炎症过程 3,6,7,8。在稳态条件下，脑膜支持许多免疫哨兵细胞，包括先天淋巴细胞（ILC），巨噬细胞，树突状细胞（DC），肥大细胞，T细胞，以及在较小程度上的B细胞9,10,11。
脑膜是高度血管化的结构，含有淋巴管，在中枢神经系统与其外围之间提供淋巴连接8,12,13,14。在由中枢神经系统损伤、感染、自身免疫甚至神经变性引起的炎症性疾病中，外周来源的免疫细胞会浸润脑实质并改变脑膜内的免疫景观。细胞浸润后，脑膜可能代表外周来源免疫细胞的功能生态位，促进免疫细胞聚集、局部免疫细胞活化和中枢神经系统区室的长期存活。在影响中枢神经系统的多种疾病中观察到明显的脑膜炎症，包括多发性硬化症 （MS）15,16,17,18,19、中风 20,21、无菌损伤 22,23（即脊髓损伤和创伤性脑损伤）、偏头痛 24 和微生物感染 25,26，27,28,29.因此，脑膜区室中常驻细胞和外周衍生免疫细胞的表征对于了解这些细胞在稳态条件和疾病发病机制中的作用至关重要。
从颅骨和椎体中提取大脑、脊髓和脑膜在技术上具有挑战性且耗时。目前还没有可用于快速提取大脑的技术，所有三个脑膜层都完好无损。虽然椎板切除术产生出色的脊髓组织形态并保留了脑膜层，但它既非常耗时又复杂30,31。相反，更传统的提取方法，例如从颅骨中取出大脑和脊髓的液压挤出有助于快速提取CNS组织，但蛛网膜和硬脑膜都因这些技术而丢失30,31。在脑和脊髓组织的常规分离过程中，硬脑膜和蛛网膜层的遗漏导致对中枢神经系统隔室内细胞的分析不完整。因此，确定专注于快速提取具有完整脑膜的CNS组织的新技术对于CNS室的最佳分析至关重要。
本手稿介绍了两种从小鼠中快速提取大脑、脊髓和脑膜的方法，有助于对中枢神经系统实质和脑膜中的常驻细胞和外周衍生免疫细胞进行下游分析。这些优化的方案侧重于 1） 分离单细胞悬液以进行下游分析和 2） 准备组织以进行组织学处理。从大脑、脊髓组织以及硬脑膜和蛛网膜脑膜32 获得单细胞悬浮液可以同时分析存在于实质和脑膜隔室中的细胞。单细胞悬浮液可用于不同的应用，包括用于体外刺激的细胞培养测定33、酶联免疫斑点（ELISpot）28、34、35、流式细胞术36、33和单细胞37或批量转录组学。此外，分别具有完整颅骨或椎体的整个大脑和脊髓脱钙的优化方案允许周围骨骼的温和脱钙，使脑膜完整并保留组织形态。该方法允许在实质和脑膜间隙内使用免疫组织化学（IHC）或原位杂交（ISH）技术选择性鉴定蛋白质或RNA。中枢神经系统内驻留细胞和外周来源免疫细胞的表型、活化状态和定位的表征可能为了解中枢神经系统区室中的单个细胞类型如何促进稳态和疾病发病机制提供必不可少的信息。

<table>
	<tbody>
		<tr>
			<td>Aluminum foil</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher Scientific</td>
			<td>37002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-12R centrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 15 mL</td>
			<td>VWR</td>
			<td>525-1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 50 mL</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Hauser Scientific</td>
			<td>5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold</td>
			<td>VWR</td>
			<td>18000-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11003-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable polystyrene tube, 14 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Fisher Scientific</td>
			<td>NC0595256</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>Airgas</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Durmont #7Forceps</td>
			<td>Fine Science Tools</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate</td>
			<td>Amresco</td>
			<td>0105-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 100%</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter top tube, 5 mL</td>
			<td>VWR</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable viability stain 780</td>
			<td>Becton Dickinson</td>
			<td>565388</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>Gallios</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Chemical</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (488 conjugate)</td>
			<td>Jackson immunoresearch</td>
			<td>115-546-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (594 conjugate)</td>
			<td>Jackson immunoresearch</td>
			<td>115-586-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit 488</td>
			<td>Jackson immunoresearch</td>
			<td>111-545-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rat 594</td>
			<td>Jackson immunoresearch</td>
			<td>112-585-167</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rat 650</td>
			<td>Jackson immunoresearch</td>
			<td>112-605-167</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balnced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Andwin Scientific</td>
			<td>02-671-51B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13004-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher chemical</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4 (anhydrous)</td>
			<td>Sigma Aldrich</td>
			<td>P5655-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Airgas</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse FC block (CD16/32)</td>
			<td>Becton Dickinson</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HP04 (anhydrous)</td>
			<td>Fisher Chemical</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher chemical</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 25 gauge</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal mouse serum</td>
			<td>ThermoFisher Scientific</td>
			<td>31881</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh strainer</td>
			<td>VWR</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 20%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713-S</td>
			<td>Diluted to 4% using 1 x PBS</td>
		</tr>
		<tr>
			<td>Pasteur pipette, 9 inch, unplugged</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Rat Anti-Mouse CD4</td>
			<td>Becton Dickinson</td>
			<td>553730</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 Rat Anti-Mouse CD19</td>
			<td>Becton Dickinson</td>
			<td>562329</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll density gradient media</td>
			<td>GE healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 Rat Anti-Mouse CD45</td>
			<td>Becton Dickinson</td>
			<td>550994</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 100 mm</td>
			<td>VWR</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher Scientific</td>
			<td>13-636-AB150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid</td>
			<td>Drummond Scientific Corporation</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 200 &#181;l</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 1 mL</td>
			<td>USA Scientific</td>
			<td>1111-2831</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 200 &#181;l</td>
			<td>USA Scientific</td>
			<td>1111-1816</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, 1 mL</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Diamond mountant with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32</td>
			<td>Becton Dickinson</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse CD3 (SP7 clone)</td>
			<td>Abcam</td>
			<td>ab16669</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse laminin</td>
			<td>Abcam</td>
			<td>ab11575</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse ERT-R7</td>
			<td>Abcam</td>
			<td>ab51824</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 1 mL</td>
			<td>VWR</td>
			<td>357521</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 10 mL</td>
			<td>VWR</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 5 mL</td>
			<td>VWR</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors, extra fine</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>Becton Dickinson</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>Becton Dickinson</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter system</td>
			<td>Millipore</td>
			<td>20207749</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum flask</td>
			<td>Thomas Scientific</td>
			<td>5340-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum in-line filter</td>
			<td>Pall Corporation</td>
			<td>4402</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum line</td>
			<td>Cole Palmer</td>
			<td>EW-06414-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>ThermoFisher Scientific</td>
			<td>Versa bath</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating central nervous system tissues and associated meninges for the downstream analysis of immune cells

中枢神经系统（CNS）由大脑和脊髓组成，被脑膜包裹，膜层是外围和CNS之间的屏障。中枢神经系统是一个免疫学特化的部位，在稳态条件下，免疫特权在中枢神经系统实质中最为明显。相比之下，脑膜含有多种常驻细胞，包括先天性和适应性免疫细胞。在由中枢神经系统损伤、自身免疫、感染甚至神经变性引发的炎症性疾病中，外周来源的免疫细胞可能会进入脑实质并居住在脑膜内。这些细胞被认为在中枢神经系统疾病发病过程中执行有益和有害作用。尽管有这些知识，但在分析中枢神经系统隔室时，脑膜经常被忽视，因为传统的中枢神经系统组织提取方法省略了脑膜层。该协议提出了两种不同的方法，用于快速分离鼠中枢神经系统组织（即脑，脊髓和脑膜），适用于通过单细胞技术，免疫组织化学和原位杂交方法进行下游分析。所描述的方法提供了对CNS组织的全面分析，非常适合评估在稳态条件下和疾病发病期间占据CNS区室的细胞的表型，功能和定位。

该代表性实验旨在量化B和T细胞，并描述B和T细胞在稳态条件下脑膜和实质CNS区室以及小鼠进行性MS模型（即TMEV-IDD）中的定位。如前所述，TMEV-IDD通过5 x 106 斑块形成单位（PFU）的TMEV BeAn的颅内感染在5周龄雌性SJL小鼠中诱导29。
本研究评估了感染后第120天慢性TMEV-IDD期间脑膜，大脑和脊髓中的B和T细胞。使用年龄匹配的假处理小鼠作为对照。该研究由两个实验组成。第一个重点是获得用于流式细胞术评估的单细胞悬液，这是一种通过评估细胞表面和/或细胞内抗原来分析和量化细胞组成的成熟技术（n = 4假处理;n = 5 TMEV-IDD）。第二个实验侧重于通过利用脱钙脑和脊髓组织的免疫组织化学来描述中枢神经系统区室中的B和T细胞定位（n = 3假处理;n = 8 TMEV-IDD）。
从TMEV-IDD和假处理小鼠的大脑，脊髓和混合脑膜（脑和脊髓）中分离出单细胞悬浮液后，对所有样品应用表面染色方案。简而言之，将单细胞悬浮液与可固定的活力排阻染色剂（780）孵育15分钟，洗涤，在小鼠血清存在下用Fc阻断封闭15分钟，并用偶联抗体染色细胞表面标志物，包括CD45（30-F11;全氯乙烯-氰酸5.5）， CD19 （1D3;PE-CF594）和CD4（GK1.5;PE）如前所述30分钟28,29。然后使用流式细胞仪28,29洗涤和分析细胞。如前所述进行活力门控28。评估CD45表达以区分CD45hi外周来源的浸润免疫细胞（P1）与CD45lo小胶质细胞（P2）和CD45-神经元，星形胶质细胞和脑和脊髓中的少突胶质细胞（P3;图3A）。在假治疗小鼠中，脊髓和脑组织中存在很少的CD45hi细胞。在脑膜中，应用与大脑和脊髓数据相同的CD45 hi表达门控截止值来识别CD45hi免疫细胞（P1;图3C）并排除脑膜中存在的非免疫细胞（即成纤维细胞、内皮细胞）。在假治疗的小鼠中，脑膜中存在很少的CD45hi细胞（<0.1%）。在TMEV-IDD CNS组织中的CD45hi免疫细胞中，B细胞和CD4 T细胞分别通过CD19和CD4的表面表达进行鉴定（图3B-D）。在所有TMEV-IDD CNS组织中，与假处理的小鼠相比，观察到CD45HI免疫细胞的百分比增加（图4A）。在慢性TMEV-IDD期间，与脑膜隔室相比，大脑和脊髓中CD45hi免疫细胞中的B细胞百分比更高（图4B）。
为了鉴定慢性TMEV-IDD期间CNS区室内B细胞和T细胞的定位，使用先前描述的染色方案使用免疫组织化学评估脱钙的大脑和脊髓29。使用层粘连蛋白（基底膜成分29,51）和ER-TR7（成纤维细胞网状细胞标志物52,53）划分脑膜和脉管系统。在从颅帽中常规提取大脑期间，软脑膜层完好无损，但排除了剩余的脑膜层（图5A）。在脊髓中，液压挤压导致所有脑膜层缺失（数据未显示）。在脱钙的大脑和脊髓中，所有脑膜层都是完整的（图5B）。为了检查慢性TMEV-IDD中的B细胞和T细胞定位，使用IgG表达来确定同种型切换B细胞29的定位，并使用CD3可视化所有T细胞29。用ER-TR7对IgG进行共染色显示，同种型切换的IgG + B细胞存在于CNS实质和脑膜中（图6A）。ER-TR7 +脑膜内的细胞聚集体包含多个IgG + B细胞和CD3 + T细胞（图6B）。
代表性结果显示，与年龄匹配的假处理小鼠相比，慢性TMEV-IDD小鼠实质和脑膜隔室中的B细胞和T细胞百分比都很高。IHC分析进一步表明，在TMEV-IDD组织中，B细胞和T细胞分散在实质中，但在脑膜中密切相关，形成炎症聚集体。在进行性 MS 患者中，脑膜炎聚集体与邻近组织损伤和更差的疾病结局有关。在慢性 TMEV-IDD 中，中枢神经系统间室中持续存在 B 细胞和 T 细胞以及脑膜中的聚集可能与组织损伤和疾病进展有关。需要进一步的研究来了解脑膜与实质炎症如何影响脱髓鞘、神经变性和临床残疾。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61166/61166fig1v2.jpg"> 图1:分离大脑和脊髓进行脱钙。 蓝色虚线表示为隔离具有完整颅骨（切口 1-4）和椎体（切口 5-8）的大脑而进行的切口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61166/61166fig1v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61166/61166fig2v2.jpg"> 图 2:分离大脑、脊髓和脑膜以进行单细胞技术。 （A）蓝色虚线表示为隔离具有完整脑膜和大脑（切口1-3）和椎体（切口5-7）的颅骨而进行的切口。侧切3在颅骨的两侧进行。（B）蓝线表示为划伤和去除颅骨盖骨中的脑膜而切开的切口，以及在椎体（两侧）上切开以隔离脊髓和脑膜的切口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61166/61166fig2v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61166/61166fig3v2.jpg"> 图 3:鉴定 CNS 组织中 CD45hi 浸润免疫细胞。（A）用于鉴定假处理（红色）或TMEV-IDD（蓝色）小鼠脊髓总活细胞中的CD45hi浸润免疫细胞（P1），CD45lo小胶质细胞（P2）和CD45-少突胶质细胞，星形胶质细胞和神经元的门控策略。在假处理的大脑和脊髓中检测到最小的CD45hi细胞。（B）用于鉴定TMEV-IDD小鼠CD45hi浸润免疫细胞（P1）中的CD19 + B细胞和CD4 + T细胞的门控策略。脑和脊髓组织的门控策略相似。（C）用于识别假处理（红色）和慢性TMEV-IDD小鼠（蓝色）脑膜中CD45hi细胞（P1）的门控策略。（D）在慢性TMEV-IDD小鼠脑膜中识别CD45hi浸润免疫细胞（P1）中CD19 + B细胞和CD4 + T细胞的门控策略。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61166/61166fig3v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61166/61166fig4v2.jpg"> 图 4:慢性 TMEV-IDD 中枢神经系统组织中 CD45hi 免疫细胞和 CD19+ B 细胞增加。从假处理或 TMEV-IDD 脑膜、脑和脊髓获得的流式细胞术数据的条形图摘要显示 CD45hi 浸润免疫细胞的百分比 （A） 或 CD19+ B 细胞的百分比 （B）。对于TMEV-IDD组织，从五只小鼠获得流式细胞术数据，分别处理脊髓和大脑，同时汇集脑膜进行分析。对于假处理的小鼠，从四只小鼠获得流式细胞术数据，分别处理脊髓和大脑，同时汇集脑膜进行分析。脊髓和大脑的数据显示为平均± SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61166/61166fig4v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61166/61166fig5v2.jpg"> 图 5:脱钙大脑和脊髓中完整的脑膜层。 （A）从颅骨帽中提取的大脑或（B）从慢性TMEV-IDD小鼠中具有完整头骨和椎柱的脱钙大脑评估DAPI（蓝色），脑膜标志物层粘连蛋白（绿色）和ER-TR7（红色）。比例尺 = 50 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61166/61166fig5v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61166/61166fig6v2.jpg"> 图6:慢性TMEV-IDD期间脑膜中的免疫细胞聚集很明显。 （A）检查慢性TMEV-IDD小鼠的脱钙化椎柱是否存在IgG（绿色），以鉴定实质和ER-TR7 +脑膜（红色）中的同种型切换免疫细胞。（B）CD3 + T细胞（绿色）和IgG +同种型转换的B细胞（红色）与ER-TR7 +脑膜共注射以评估脊髓组织内的定位。白色箭头突出显示脑膜内的 B 细胞和 T 细胞。比例尺 = 50 μm。白框描绘了为裁剪图像选择的区域（右;比例尺= 10 μm）。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61166/61166fig6v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells at JoVE.com

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

本文提出了两种优化的方案，用于检查中枢神经系统内的常驻和外周来源的免疫细胞，包括大脑、脊髓和脑膜。这些协议中的每一个都有助于确定在稳态和炎症条件下占据这些隔室的细胞的功能和组成。

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

isolating-central-nervous-system-tissues-associated-meninges-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.9K 次观看.  Dartmouth-Hitchcock Medical Center. 本文提出了两种优化的方案，用于检查中枢神经系统内的常驻和外周来源的免疫细胞，包括大脑、脊髓和脑膜。这些协议中的每一个都有助于确定在稳态和炎症条件下占据这些隔室的细胞的功能和组成。

视频: Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

在鼻内siRNA输送到中枢神经系统期间放置小鼠的定位装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...