我们感谢SusanneSørensen的技术帮助。这项工作得到了Velux基金会的资助;茉莉花基金会Aase和Ejnar Danielsens基金会;哈特曼兄弟基金会导演Jacob Madsen和妻子Olga Madsens基金会;医生Sofus Carl Emil Friis和妻子Doris Friis的信任; 1870年的基础; Torben和爱丽丝Frimodts基金会和神经研究基金会。手稿编辑由英格伍德生物医学编辑执行。

所有动物手术均按照丹麦动物实验检验局的指导方针进行，并由当地实验动物伦理委员会批准。 组织加工注意:在4℃下在0.2M磷酸盐缓冲液中稀释的4％多聚甲醛（PFA）中的经心内灌注和固定后，将大脑在4℃下转移到磷酸盐缓冲盐水（PBS）中30％蔗糖三天。此后，将大脑冷冻在粉末状干冰上，并储存在-80℃直到切片。 <ol><li>使用手术刀沿着大脑中线分开右半球和左半球，然后在第一脑中随机选择一个或另一个半球，然后在右半球和左半球之间系统地移动（ 图1A ）。将采样的半球安装在垂直于光盘的长轴的样品盘上使用安装介质。 </li><li>放置编号多个容器用于在低温恒温器中进行预冷。 </li><li>将样品盘放入低温恒温器中，并将整个海马（伯尔玛1.80至7.0416）切割成冠状平面内的80μm厚的切片（ 图1B ）。 <ol><li>将所有取样部分连续放置在冷却的多容器中，以确保切片保持切割顺序。 </li><li>用冷冻保护剂完全覆盖部分，并将容器保持在-20°C直到进一步使用。 </li></ol></li></ol> 2.免疫染色注意:要在整个染色程序中跟踪各个部分，将采样部分置于25孔染色网中。每5 个部分使用，每个海马平均最终数量为12（8-16）个，用于免疫染色和随后的细胞计数。 <ol><li>在第n节和第n节（ 图1C ）之间随机开始进行二次采样之前，将容器从-20°C转移到室温（RT）。 </li><li>使用例如画笔将数字顺序的部分转移到放置在装有PBS的培养皿中的25孔染色网。 注意:从这一点上，将染色网中的部分放置在放置在定位摇床上的匹配玻璃盘中（2.3至2.9节）。 </li><li>将染色网转移到匹配的玻璃皿中，然后在稀释在蒸馏水（dH 2 O）中的3％过氧化氢（H 2 O 2 ）中孵育20分钟后，在室温下在PBS中洗涤两次10分钟。 </li><li>将切片转移到三种不同的盐酸（HCl）溶液（1℃，0℃，10分钟，2分钟，室温，10分钟，2分钟，37℃，20分钟），然后中和0.1M四硼酸钠一个t RT 20分钟。 </li><li>在用PBS（洗涤缓冲液）稀释的1％Triton X-100中孵育3×10分钟后，将切片转移到含有10％胎牛血清（封闭溶液）的洗涤缓冲液中1小时。 </li><li>在4℃下，在封闭溶液中以1:100稀释的小鼠抗BrdU孵育切片48小时。 </li><li>在洗涤缓冲液中洗涤3×10分钟，并在洗涤缓冲液中以1:10稀释的辣根过氧化物酶孵育48小时。 </li><li>将其转移到PBS中5×10分钟，然后在0.01％二氨基联苯胺（DAB）中稀释7分钟，然后将其转移到含有0.02％H 2 O 2的类似DAB溶液中，在室温下10分钟。 </li><li>完成一系列洗涤（PBS中2×10分钟，磷酸盐缓冲液不加氯化钠2×10分钟），并在显微镜载玻片上以数字顺序安装。 </li><li>将切片干燥约30分钟，然后用甲酚复染紫色。 </li><li>将显微镜载玻片放在滑架上。 </li><li>在含有dH 2 O的载玻片染色皿中将切片再水合10分钟，然后将载玻片转移到dH 2 O中的0.02％甲酚紫15分钟。 </li><li>重复步骤2.12，然后将切片在乙醇中脱水三次（96％，5分钟，99％，2×2分钟）。 </li><li>将切片置于二甲苯中2×15分钟，并使用快速干燥介质将载玻片覆盖滑动。 </li><li>最后，将盖子滑动的玻片留下大约24小时，然后才能用于显微镜。 </li></ol> 3.使用光学分馏器估计BrdU标记的神经元总数注意:进行包括几只动物在内的初步研究，以确定最佳采样参数，例如要分析的部分数量和采样部分内的光学分离器数量。这个飞行员也提供了一个初步CV（标准偏差/平均值）和调整CE的可能性（见第3.9.3节），以获得调查员确定的估计精度（详见下文）。同样进行z分布分析以解决以下几点:组织收缩，整个部分染色的质量和细胞在z轴上的分布14 。 <ol><li>检查部分中组织的厚度，以确认它们适用于使用光学分馏器， 例如 ，对于所选择的高度（包括防护区域），足够厚度（见下文）。注意:组织厚度是感兴趣区域内几个地方的措施。 <ol><li>将幻灯片放置在显微镜的电动平台上，并打开首选的立体软件。 </li><li>使用低放大倍数目标（2X或4X），在切换到100X oil-imm之前描绘感兴趣的区域（ 见图2A ）。 </li><li>使用计数框的特定点（ 例如在拐角处或与之相邻），通过沿着焦平面移动来定位该部分的顶部，直到该部分的某些特征出现焦点。将此z位置注册为0（ 见图2B ）。 </li><li>将焦平面向下移动通过组织，直到计数框的相同特定点处于聚焦组织的最后z级，并标记此位置。局部组织厚度从0到该端点定义，并且可以在z轴上读取。注册组织厚度（ 见图2B ）。 </li></ol></li><li>记录细胞在整个部分的厚度上的染色和分布的完全渗透。 注意:BrdU标记的神经元的分布被用作确定所需保护区的指南。尽管细胞的均匀分布至关重要在该部分的顶部和底部附近观察到更少的细胞是可以接受的，这表明失去了上限的影响（进一步的细节见Dorph-Petersen等人，2009）。 <ol><li>样品BrdU标记的神经元，并将z位置与每个计数框的选定角度中测量的局部区域厚度一起进行记录。 </li><li>绘制BrdU阳性神经元的数量作为其z位置的函数。 </li></ol></li><li>根据平均切片厚度和细胞分布，确定分离器和防护区的高度（见3.1和3.2）。 注意:分离器的高度应小于截面厚度，以避免靠近表面的伪影。通过将防护区包括在该部分的顶部和底部来避免这种风险。 </li><li>确定分隔符之间的步长。 <ol><li>描述所有要分析的部分的兴趣区域，并注册该区域。 </li><li>总结这些区域，然后划分b就是这个地区的职业人数。 注意:根据以往的经验，一个良好的起点需要75个探针。这将提供面积和相应采样位置的近似值（A 步 ）（更多详情，请参阅西方，2012年）。 </li><li>取A 步的平方根，将提供xy步长。 </li></ol></li><li>确定计数框的大小。 <ol><li>将计数帧的大小设置为任意单位，并使用第3.3和3.4节中获得的参数对BrdU阳性神经元进行导频采样。 注意:尺寸必须通过试验和错误凭经验来确定，但建议调整其尺寸以使每个隔离层8采样2-3个细胞。 </li></ol></li><li>在试点研究的最后一步开始进行细胞抽样。 注意:获得的采样参数应导致大约150-200个单元i的计数每个动物足以获得有效的立体设计8 。 </li><li>使用100×油浸物镜计算BrdU阳性神经元，最终放大倍数为2000-3000×。在每个光学检测器中，识别由感兴趣特征明确识别的任何BrdU标记的神经元（在本研究中，标准是当BrdU标记的神经元的前沿首次成为焦点时），位于在计数框内或触摸夹杂线（ 图2B ）。不要计数BrdU阳性神经元，当被感染高度的特征清楚地识别时，触摸排除线。在整个立体定量化过程中严格遵守这些计数规则至关重要。 注意:由于BrdU并入所有分裂细胞，形态学被用于区分神经元和其他类型的细胞。 NEUron特征是具有中性细胞质和暗核仁的明确定义的核。与神经胶质细胞相比，BrdU阳性细胞基于其相对较大的尺寸被认为是新形成的神经元。应该提到的是，在短期增殖研究（例如&lt;24 h）中，使用未成熟神经元标记的双染可能是先决条件17 。 </li><li>通过将计数的总细胞数（ΣQ - ）与倒数采样分数相乘来估计每个脑中BrdU阳性神经元的总数: <img alt="方程" src="/files/ftp_upload/55737/55737eq1.jpg" /> 对于<img alt="方程" src="/files/ftp_upload/55737/55737eq2.jpg" /> 哪里<img alt="方程" src="/files/ftp_upload/55737/55737eq3.jpg" /> Q-是加权平均截面厚度， h是分界器的高度， asf是面积采样分数， ssf是截面s放大分数，t i是具有细胞计数的第 i 个计数帧中的截面厚度<img alt="方程" src="/files/ftp_upload/55737/55737eq4.jpg" />在分区14,18中 。 </li><li>计算系统均匀随机抽样（SURS）中BrdU阳性神经元抽样方差的误差系数（CE） 8 。 <ol><li>首先计算噪声 ，等于计数的BrdU阳性神经元总数: <img alt="方程" src="/files/ftp_upload/55737/55737eq5.jpg" /></li><li>接下来，计算SURS中的差异: 注意:我们已经判断海马区域和细胞计数从一个部分到另一个平滑变化，因此使用"平滑度等级"m = 1（1/240）8,19 <img alt="方程" src="/files/ftp_upload/55737/55737eq6.jpg" /> 哪里 <img alt="方程" src="/files/ftp_upload/55737/55737eq7.jpg" /> <img alt="方程" src="/files/ftp_upload/55737/55737eq8.jpg" /> <img alt="方程" src="/files/ftp_upload/55737/55737eq9.jpg" /> P i是第i部分中对象的点数，n是部分4,20的数量。 </li><li>最后，计算估计的CE: 注意:通常，当CE值小于观察到的CV的一半时，典型地实现最佳精度，如OCV 2 = ICV 2 + CE 2 ，其中OCV是观察到的CV，而ICV是固有的CV 4 。 <img alt="方程" src="/files/ftp_upload/55737/55737eq10.jpg" /></li></ol></li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/55737/55737fig1.jpg" /> F图1:根据本研究中使用的分馏器原理显示组织处理的示意图。 经实验，半球分离，右半球或左半球系统地随机选择（A）。将整个海马延伸的完整脑切成80μm厚的冠状切片（B），于是每5 个部分被亚采样，从第1至第5部分之间随机开始，用于最终的免疫染色和立体定量（C）。 <a href="http://ecsource.jove.com/files/ftp_upload/55737/55737fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55737/55737fig2.jpg" /> 图2:使用光学隔离器的立体取样程序的图示。 （A）组织学处理后海马GCL / SGZ是该区域的均匀随机应用的描绘和光学隔离。 （B）在光学障碍物（左）中，仅当感兴趣的特征在分辨率高度内被清楚地识别出并且位于计数框架内部或接触包围线（右侧）时才计数BrdU阳性神经元。触摸排除线的神经元不算。刻度棒=10μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55737/55737fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereology:+the+fast+lane+between+neuroanatomy+and+brain+function--or+still+only+a+tightrope?.">Stereology: the fast lane between neuroanatomy and brain function--or still only a tightrope?.</a> Acta Neurol. Scand. Suppl. 137, 8-13 (1992).</li></ol>

作者没有竞争的经济利益。

使用ECS作为诱导神经发生的方法，我们发现海马中新的BrdU阳性神经元的形成立即增加了260％。在这个急性产生的神经元池中，我们发现从第1天到第三个月有40％的磨损，近10％的新形成的神经元在治疗后至少12个月存活10,11 。 BrdU标记的神经元的计数遵循严格的采样方案，其中整个海马被切成80μm厚的部分，然后每第 5 个部分进行亚采样，随机抽样开始于第一部分和第五部分之间。提供这些部分是以系统随机的方式进行选择的，这显然是减少最终结果的差异的极好方法，没有详尽的计数1 。该采样方案允许我们平均计数12（8-16）个海马中的BrdU阳性神经元每个大鼠脑中的切片，最终精度为9-11％。 当使用分馏器方法时，必须知道进行计数的区段高度的分数，因为组织收缩和变形在组织学处理期间经常发生14 。实际上，我们在这项研究中看到了厚度的大幅收缩。此外，应该注意的是，可能会发生差异组织收缩，如Dorph-Petersen等人然而，只要感兴趣的完整结构可用于分析，这些限制不会导致颗粒总数即 BrdU阳性神经元的偏差。在组织收缩是一个特殊问题的研究中，应该总是指出，结果是在变形的组织中获得的。在这项研究中，我们计算出一个10毫米的检测器高度。本研究的部分最终平均厚度为26μm，为5μm顶部的防护区域和该部分底部的11-μm（平均）防护区域。这些参数是可以接受的，因为防护区域应近似为采样颗粒的直径。 在划定GCL / SGZ后，各界人士均匀地随机放置在划定区域内。分隔器应以固定的步长定位，优化采样和计数，以有效地获得由调查员确定的精度的估计。通常通过在感兴趣的结构5中计数多达150-200个细胞来实现最佳精度。在本研究中，我们计算了每只大鼠海马中平均133（33-372）BrdU阳性神经元。由于一些对照动物中的细胞数量较少，我们的BrdU阳性神经元计数低于获得合适精度所需的细胞数量，这导致了相对较高的CE值。 H不过，由于我们获得的CV值低于CV值的一半（见代表性结果部分），因此不需要额外的抽样和计数。高CV值表明观察到的变化的最大贡献来源于生物学变化。实际上，我们可能认为本研究中计数的BrdU阳性神经元的平均数量高于必需。例如，在一组老鼠中，我们获得了9％的CE值，观察到CV值为43％。在这种特殊情况下，我们可以瞄准精度约20％。总之，本研究中BrdU阳性神经元总数的精确度足以证明实际治疗效果对真实粒子数的影响。由于某些动物群体的相当大的生物学变化，可以以可接受的精度获得相同的估计，尽管努力的支出更少。组内只有高生物差异通过增加动物的数量来补偿。 人们可能会认为，获得精确细胞数量的黄金标准需要穷举所有感兴趣的物体。然而，在大脑的大多数研究中，这不是由于大量细胞的可能性。尽管比穷尽细胞计数效率更高，但是与单细胞数差异的简单筛选相比，任选的分馏器相对来说是相当耗时的，如果精确数目的细胞不是必需的，则是优选的。根据我们的经验，纯粹通过筛选程序可以检测到超过20-30％的差异。 如果正确执行，使用基于设计的体视学进行采样可以有效地提供无偏差和精确的估计1 。体现的不偏不倚的属性产生的估计，当被复制时，近似真实的群体平均值，并提高重现性估计21 。最初，必须获得感兴趣的整个结构的代表性样本（在本研究中是海胆GCL / SGZ），允许在第4节的统计学有效子集中进行估计。为了获得适当数量的部分和计数探针，我们应用SURS，与随机抽样相比减少方差8 。此外，SURS确保结构内的感兴趣的粒子以相同的概率进行采样，而与结构中的尺寸，形状，取向和分布无关。在获得精确和无偏估计时，强烈推荐这样的立体观点是项目的核心方面。

三维（3-D）结构中的细胞的定量可能需要基于无偏的原理获得估计。即使在今天，研究也经常使用二维（2-D）方法来报告三维结构的数据。然而，这些方法在细胞的形状和分布方面不考虑结构的异质性，导致有限的限制;他们对3-D兴趣结构做出假设，结果并不是指完整的结构。这些限制降低了方法的灵敏度和准确性，并增加了错误的风险1 。 细胞计数中的偏差可以通过应用基于设计的立体学来避免，这对于获得关于给定组织或结构中的细胞数量的定量信息是非常重要的。 虽然立体学以前是一个非常耗时的方法，程序的进步，软器具和成像，使其变得更加高效和广泛适用。立体学需要统计抽样原理和随机几何理论，以提供有效的工具，用于通过2维部分2中的抽样来估计三维结构中的体积，表面积，长度和物体数量。因此，立体学允许人们获得关于组织切片结构变化的无偏量化数据，这些数据给出了真实数据的平均估计，无需详尽的分析1 。立体学定义为采样信息中几何参数的统计推断。这可以通过公正的抽样来实现，也就是说系统的随机抽样，以及计数规则，确保所有对象具有相等的概率被计算3 。虽然立体结果可能具有不同程度的精度，如误差系数（CE）所示，这可以是广告通过根据需要调整采样量4,5 ，适合于固有生物方差（方差系数（CV））。以前的几个立体学研究已经研究了电惊厥刺激（ECS）对啮齿动物的海马可塑性的短期影响。来自这些研究的数据显示，在重复的ECS之后神经元总数以及突触分化升高的初始增加。然而，这些研究不直接估计神经发生，因为它们不使用特异性标志物进行细胞增殖。 在这里，我们提出一种立体方法的应用，光学分级技术8,9用于量化大鼠海马颗粒细胞层（GCL）中新形成的神经元的总数，包括亚颗粒区（SGZ）以及作为他们的长期urvival 10，11 。我们对大鼠进行ECS临床相关时间表（每周三次，持续3周），这是神经发生最有效的刺激物之一12 。使用相同的程序治疗对照动物，但不通过电流。在21天的实验中，在细胞分裂期间，将所有大鼠腹膜内注射5-溴-2&#39;-脱氧尿苷（BrdU），其中并入DNA以代替胸苷。实验后，将大鼠保持四个不同的时间段（24小时，3个月，6个月和12个月）。使用分馏塔原理，我们通过对各部分进行均匀随机取样并将光学隔离器（3-D探针）应用于采样和免疫染色的厚组织切片，从而获得海马的已知部分。特别值得注意的是，冻结部分通常在加工过程中在z轴上塌陷在这种特殊情况下应使用基于加权平均截面厚度的光学隔离层14 。当光学障碍物的焦平面向下移动已知距离时，BrdU阳性神经元被计数，当感兴趣的特征被清楚地识别。在立体学领域，有一些关于应该计算的粒子总数的争论15 ;通常在感兴趣的结构中计算150-200个神经元以获得足够的精度， 即 7-8％的系数8 。 BrdU阳性神经元计数使用具有摄像机的标准显微镜成像的立体软件完成，并且具有以下目的:2X，4X和10X以及100X油浸物镜（数值孔径= 1.40），以及电动阶段。用数字微量扫描器测量z方向的运动。最终放大倍数是3000X。

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Materials&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>5&#039;-bromo-2&#039;deoxyuridine - BrdU</td><td>Sigma-Aldrich</td><td>19-160</td><td></td></tr><tr><td>Cresyl Violet</td><td>Sigma-Aldrich</td><td>C5042</td><td>Contains the following materials for 1 L: 0.2 g cresyl violet; 2.0 g sodiumacetate, 3 H2O; 3 ml glacial acetic acid; 1000 ml destilled water&amp;nbsp;</td></tr><tr><td>Cryoprotectant</td><td>Capital Region Pharmacy, DK</td><td>861334</td><td>Contains the following materials: Sucrose; polyvinylpyrrolidon PVP 40; sodiumphosphatebuffer (0.2 M);&amp;nbsp; ethylenglycol; demineralized water.</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Capital Region Pharmacy, DK</td><td>817205</td><td></td></tr><tr><td>Diaminobenzidine &amp;ndash; DAB</td><td>Sigma-Aldrich</td><td>D5637</td><td></td></tr><tr><td>Envision-HRP</td><td>DAKO</td><td>K4001</td><td>Peroxidase labeled polymer conjugated to goat anti-mouse immunoglobulins</td></tr><tr><td>Ethanol - 70 %</td><td>Plum A/S</td><td>201098</td><td></td></tr><tr><td>Ethanol - 96 %</td><td>Plum A/S</td><td>201104</td><td></td></tr><tr><td>Ethanol - 99.9 %</td><td>Plum A/S</td><td>201111</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>In Vitro</td><td>BI-04-003-1A</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 30%</td><td>Capital Region Pharmacy, DK</td><td>896456</td><td></td></tr><tr><td>HCl</td><td>J. T. Baker</td><td>6081</td><td></td></tr><tr><td>Mounting medium</td><td>Sakura</td><td>4583</td><td>Tissue Tek</td></tr><tr><td>Mouse-anti-BrdU</td><td>Becton-Dickinson</td><td>347580</td><td></td></tr><tr><td>PBS buffer</td><td>Capital Region Pharmacy, DK</td><td>853436</td><td>pH = 7.4</td></tr><tr><td>PFA</td><td>VWR</td><td>28,794,295</td><td>pH = 7.4</td></tr><tr><td>Phosphate buffer&amp;nbsp;</td><td>Capital Region Pharmacy, DK</td><td>866533</td><td>0.1 mol/l, pH 7.4</td></tr><tr><td>Rapid drying mounting medium</td><td>Histolab Products AB</td><td>801</td><td>Pertex</td></tr><tr><td>Sodium Tetraborate</td><td>Merck A/S</td><td>1,063,080,500</td><td></td></tr><tr><td>Sucrose</td><td>Merck A/S</td><td>1,076,515,000</td><td></td></tr><tr><td>Triton X-100</td><td>Merck A/S</td><td>1,086,031,000</td><td></td></tr><tr><td>Xylene</td><td>VWR</td><td>28,973,363</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Equipment&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cover slips</td><td>Menzel-Gl&amp;auml;ser</td><td>24x50 mm #0</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>&amp;nbsp;CM1860</td><td></td></tr><tr><td>Digital Microcator</td><td>Heidenhain</td><td>VRZ 401</td><td></td></tr><tr><td>Glass dishes</td><td>Brain Resaerch Laboratories</td><td>1256</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse 80i</td><td></td></tr><tr><td>Microscope camera</td><td>Olympus</td><td>DP72</td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>48311-703</td><td>SuperFrost+</td></tr><tr><td>Motorized stage</td><td>Prior</td><td>H101A</td><td></td></tr><tr><td>Multidish Containers</td><td>Sigma-Aldrich</td><td>D6315-1CS</td><td>&amp;nbsp;Nuclon 12-wells</td></tr><tr><td>New-Cast software</td><td>Visiopharm</td><td>ver. 4.5.6.440</td><td></td></tr><tr><td>Objective 2x</td><td>Nikon</td><td>CFI Plan UW 2X</td><td></td></tr><tr><td>Objective 4x</td><td>Nikon</td><td>CFI Plan Fluor 4X</td><td></td></tr><tr><td>Objective 10x</td><td>Nikon</td><td>CFI Plan Fluor 10X</td><td></td></tr><tr><td>Objective 100x oil immersion</td><td>Nikon</td><td>CFI Plan Apo Lambda DM 100X Oil</td><td>Numerical aperture 1.40</td></tr><tr><td>Orbital shaker</td><td>Gemini BV</td><td>CM-9</td><td>Sarstedt</td></tr><tr><td>Slide rack</td><td>Sakura</td><td>4465</td><td></td></tr><tr><td>Slide staining dishes</td><td>Sakura</td><td>4457</td><td>Tissue-Tek</td></tr><tr><td>Specimen disc</td><td>Leica</td><td>14037008637</td><td></td></tr><tr><td>Staining nets</td><td>Brain Research Laboratories</td><td>2512</td><td>25-wells</td></tr></tbody></table>

the optical fractionator technique to estimate cell numbers in a rat model of electroconvulsive therapy

设计立体方法来描述定量参数，而不对细胞或结构的大小，形状，取向和分布进行假设。这些方法对于哺乳动物脑的定量分析是革命性的，其中体积细胞群体太高而不能手动计数，立体定向是当需要从二维测量中提取三维量的估计时的选择技术部分。所有的立体观念原则上都是无偏见的;然而，它们依赖于关于感兴趣结构和组织特征的适当知识。立体学是基于系统统一随机抽样（SURS），将抽样调整到精确度最有效的水平，提供关于整个兴趣结构的可靠的定量信息。在这里，我们提供光学分级器与BrdU免疫组织化学t结合o估计电惊厥刺激后大鼠海马颗粒细胞层（包括亚颗粒区）中新形成的神经元的生成和存活，这是神经发生最有效的刺激物。光学分馏器技术被设计为提供从全部结构采样的厚部分中的细胞总数的估计。厚部分提供了观察细胞在其3-D范围内的机会，从而允许基于形态学标准的容易和鲁棒的细胞分类。当正确实施时，光学分馏器的灵敏度和效率提供了固定和预定精度的精确估计。

抽样参数通过分析试验研究中的z-分布（ 图3 ），我们发现BrdU阳性神经元在分泌物高度上的均匀分布。测量截面的收缩率;最终平均厚度为最初切割为80μm的截面为26.4μm（19.0-32.0μm）。根据BrdU标记神经元的组织厚度和分布，将检测器高度设定为10μm，将截面顶部和底部的保护区分别设定为5μm和4-17μm。取决于单元格的密度，计数框的面积为1414或5210μm2。在x和y方向上的步长为220μm，并且平均每只动物12次（范围8-16）切片进行细胞计数。这些采样参数导致平均值为133（范围33-372）BrdU在每个脑半球中，173个（107-204）个间隔区的阳性细胞计数。 例如，我们在ECS治疗的大鼠中获得了BrdU阳性神经元估计的以下CV和CE值:24小时:CV = 0.59，CE = 0.11; 3个月:CV = 0.34，CE = 0.12; 6个月:CV = 0.43，CE = 0.09; 12个月:CV = 0.22，CE = 0.09。 我们使用光学分级器结合BrdU染色技术10,11量化了在ECS后大鼠海马中新形成的神经元的总数及其长期存活。最终结果显示四组对照动物的基底神经发生，其作为生存时间的函数没有显着差异（ 图3 ）。此外，ECS在24天引起BrdU阳性神经元总数的显着增加与对照动物相比，实验后小时（259％，p &lt;0.0001），3（229％，p &lt;0.001），6（152％，p &lt;0.05）和12个月（162％，p &lt; <img alt="图3" class="xfigimg" src="/files/ftp_upload/55737/55737fig3.jpg" /> 图3:大鼠海马GCL / SGZ中BrdU阳性神经元总数的定量数据10,11 。 水平条表示平均值。缩写:GCL，颗粒细胞层; SGZ，亚颗粒带;小时m，几个月。 **** p &lt;0.0001，*** p &lt;0.001和* p &lt;0.05与对照组（n = 11-12 /组）。 <a href="http://ecsource.jove.com/files/ftp_upload/55737/55737fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy at JoVE.com

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

在这里，我们提出了一种立体学方法，光学分级器，用于量化新的神经元的形成，以及它们的生存，在大脑海马中的电惊厥刺激。当正确实施时，立体感方法的灵敏度和效率确保了具有固定和预定精度的精确估计。

用于估计电惊厥治疗大鼠模型中细胞数的光学分级技术

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

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11.6K 次观看.  Bispebjerg-Frederiksberg Hospital. 在这里，我们提出了一种立体学方法，光学分级器，用于量化新的神经元的形成，以及它们的生存，在大脑海马中的电惊厥刺激。当正确实施时，立体感方法的灵敏度和效率确保了具有固定和预定精度的精确估计。 

视频: The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

颅放疗引起的认知功能障碍恢复干细胞移植策略

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

科研

医学

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

神经科学

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

大鼠电癫痫发作与海马的分离检测突触后密度蛋白的变化

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

用光学分馏塔和标准显微镜设备对小鼠黑质多巴胺能神经元数的视学估计

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

使用双边子宫内电渗透的管道，以询问遗传对啮齿动物行为的影响

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

生物学

Intermittent Photic Stimulation in Rabbit Model: A Technique to Induce Epileptic Seizures and Simultaneously Record Multisystem Parameters

For photic stimulation, place a light source with a circular reflector 30 centimeters in front of the rabbit at eye level, with the flash intensity set to the maximum, and place two mirrors on each side of the head, and one mirror behind the rabbit so that the light enters the rabbit&#39;s eyes. Attach the light to a controller with an adjustable rate, intensity, and duration.
With the photic stimulator set to 1 Hertz and the flash on, record the response for 30 seconds. Cover the rabbit&#39;s eyes with a mask to simulate or cause eye closure and stimulate for another 30 seconds. After recording the eyes-open and eyes-closed response at each frequency, turn off the photic stimulator for 30 seconds and set the controller to the next frequency setting.
After recording for 60 seconds at each frequency from 1 to 25 Hertz in 2 Hertz intervals, decrease the frequency from 60 to 25 Hertz in 5 Hertz increments and record for 30 seconds with the eyes open and 30 seconds with the eyes closed. At the end of the experiment, remove the EEG and ECG leads from the rabbit and return the animals to their home cages for routine care by the husbandry staff.