该研究得到了国家自然科学基金（81960226,81760223）的资助;云南省自然科学基金（202001AS070045,202301AY070001-011）

该实验方案已获得昆明医科大学动物伦理与使用委员会（kmmu20220945）的批准。本研究使用Sprague-Dawley（SD）两性大鼠，300-350g。
1. 诱导SAH模型
<ol><li>使用2%异氟烷和100%氧气麻醉大鼠。通过提供异氟烷（1%-3%）的连续吸入麻醉来维持麻醉。使用立体定位装置固定大鼠的头部（参见 材料表）。</li>
	<li>按照以下步骤创建 SAH 模型。<ol><li>将显微注射针插入鞍上蓄水池。然后，使用注射泵将来自大鼠尾动脉（不含抗凝剂）的自体血液注入鞍上池（参见 材料表）。</li>
		<li>使用上述坐标将显微注射针植入右侧脑室:前囟，-0.8 mm;横向，1.4毫米;深度，4 mm9。对于 SAH 组，注射 0.2 mL 大鼠尾动脉血。对于假组，施用相同体积的等渗盐水（图1A）。</li>
	</ol></li>
</ol>2. 脑切片的制备和稳定
<ol><li>使用2%异氟烷吸入深度麻醉大鼠。斩首后，取出整个大脑并将它们浸入冰冷的人工脑脊液 （ACSF） 中。 注:使用具有以下成分的新鲜制备的 ACSF 溶液:134 mM NaCl、2.8 mM KCl、29 mM NaHCO3、1.1 mM NaH 2 PO 4、1.5 mM MgSO4、2.5 mM CaCl 2和 10.11 mM D-葡萄糖（参见材料表）。将 pH 值保持在 7.3 到 7.4 之间。</li>
	<li>使用振动切片机（参见材料表）在用5%CO2和95%O 2充气的冰冷ACSF中制备厚度为200μm的SD大鼠的急性脑切片（图2）。</li>
	<li>将 200 μm 厚的急性脑切片放入浸没在 ACSF 中的尼龙网的储存室中。用95%O 2和5%CO2平衡ACSF溶液，保持35-37°C的温度范围。 让脑切片稳定2小时，让它们有时间调整（图3）。</li>
</ol>3. 用 TO-PRO-3 标记急性脑切片中的周细胞
注:使用示踪剂 TO-PRO-310 荧光标记来自急性脑切片的周细胞。
<ol><li>将荧光染料TO-PRO-3加入ACSF中，达到1μM的终浓度。 将急性脑切片在室温下与含有TO-PRO-3的ACSF在黑暗环境中孵育20分钟。 注意: 请记住在处理 TO-PRO-3 时戴上乳胶手套以提供保护。</li>
	<li>将携带脑切片的尼龙网过滤器从装载室转移到 6 孔板冲洗室（参见 材料表）。让脑切片在冲洗室中停留10分钟（图4D）。 注意:此冲洗步骤终止染色过程，减少背景标记，并防止非特异性染色。</li>
	<li>使用与 5% CO 2 和 95% O2 的混合物平衡的 ACSF 溶液冲洗制剂共 15 分钟。该冲洗步骤可阻止染料吸收并最大限度地减少背景标记（图4C）。</li>
</ol>4.急性脑切片中脑微循环的非重要周细胞染色
<ol><li>在黑暗环境中，将预装有TO-PRO-3的脑切片与Alexa Fluor 488（FITC-ISOB4;5μg/ mL，参见 材料表）偶联的异凝集素B4在37°C孵育30分钟。孵育后，在ACSF中冲洗脑切片15分钟（图4E）。</li>
	<li>像往常一样，在用 5% CO 2 和 95% O2 充气的 ACSF 中孵育脑切片。在37°C下将浓度为37μM的碘化丙啶（PI）加入两种溶液中。 这将标记非重要的脑周细胞。将制剂在该ACSF溶液中在黑暗中孵育60分钟（图4F）。</li>
	<li>接下来，用ACSF溶液冲洗制剂15分钟，以阻止染料吸收并最大限度地减少背景标记。</li>
</ol>5. 急性脑切片中重要和非重要脑周细胞的成像
<ol><li>使用塑料巴斯德移液管轻轻地将脑切片转移到玻璃底共聚焦培养皿（见材料表）上。用先前用 5% CO 2 和 95% O2 平衡的 ACSF 溶液填充培养皿。使用铁网将大鼠脑切片固定到位。</li>
	<li>将玻璃底共聚焦培养皿转移到共聚焦显微镜的载物台上。将急性脑切片放置在培养皿底部，并在共聚焦显微镜成像期间用95%O 2和5%CO2的混合物平衡的ACSF溶液灌注9（图5A和图5Ab'）。</li>
	<li>使用 40 倍物镜可视化大脑皮质微血管系统的壁细胞。使用兼容软件捕获图像堆栈（参见 材料表）。对IB4（激发/发射460nm/520nm）、PI（激发/发射545nm/595nm）和TO-PRO-3（激发/发射606nm/666nm）使用适当的滤光片（图5D）。 注意:使用以下详细信息捕获图像:40×DIC N1 物镜，3 × 12 位:512 × 512 像素（0.22 × 0.22 mm），校准:0.42 μm/px。</li>
	<li>根据脑微血管系统和周细胞的网络和形态识别它们。在每个脑切片上找到一个包含脑微血管系统网络的特定区域并捕获图像。</li>
	<li>仔细记录成像位置，以确保后续捕获的一致性（图5B，C）。如需进一步处理和分析，请使用图像分析软件（ImageJ）和照片编辑软件（参见材料表）。</li>
</ol>

蛛网膜下腔出血后脑周细胞收缩力的可视化

<ol>
	<li>Dalkara, T., Gursoy-Ozdemir, Y., Yemisci, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+microvascular+pericytes+in+health+and+disease.">Brain microvascular pericytes in health and disease.</a> Acta Neuropathologica. 122 (1), 1-9 (2011).</li><li>Dore-Duffy, P., Cleary, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+properties+of+pericytes.">Morphology and properties of pericytes.</a> Methods in Molecular Biology (Clifton, N.J). 686, 49-68 (2011).</li><li>Peppiatt, C. M., Howarth, C., Mobbs, P., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+control+of+CNS+capillary+diameter+by+pericytes.">Bidirectional control of CNS capillary diameter by pericytes.</a> Nature. 443 (7112), 700-704 (2006).</li><li>Attwell, D., Mishra, A., Hall, C. N., O'Farrell, F. M., Dalkara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+a+pericyte.">What is a pericyte.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 36 (2), 451-455 (2016).</li><li>Yemisci, M., Gursoy-Ozdemir, Y., Vural, A., Can, A., Topalkara, K., Dalkara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+contraction+induced+by+oxidative-nitrative+stress+impairs+capillary+reflow+despite+successful+opening+of+an+occluded+cerebral+artery.">Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery.</a> Nature Medicine. 15 (9), 1031-1037 (2009).</li><li>Korte, N., 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=Noradrenaline+released+from+locus+coeruleus+axons+contracts+cerebral+capillary+pericytes+via+&#945;2+adrenergic+receptors.">Noradrenaline released from locus coeruleus axons contracts cerebral capillary pericytes via &#945;2 adrenergic receptors.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. , (2023).</li><li>Hirunpattarasilp, C., Barkaway, A., Davis, H., Pfeiffer, T., Sethi, H., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxia+evokes+pericyte-mediated+capillary+constriction.">Hyperoxia evokes pericyte-mediated capillary constriction.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 42 (11), 2032-2047 (2022).</li><li>Neuhaus, A. A., Couch, Y., Sutherland, B. A., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+to+study+pericyte+contractility+and+responses+to+ischaemia+in+vitro+using+electrical+impedance.">Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 37 (6), 2013-2024 (2017).</li><li>Gong, Y., 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=Increased+TRPM4+Activity+in+cerebral+artery+myocytes+contributes+to+cerebral+blood+flow+reduction+after+subarachnoid+hemorrhage+in+rats.">Increased TRPM4 Activity in cerebral artery myocytes contributes to cerebral blood flow reduction after subarachnoid hemorrhage in rats.</a> Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics. 16 (3), 901-911 (2019).</li><li>Mai-Morente, S. P., 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=Pericyte+mapping+in+cerebral+slices+with+the+far-red+fluorophore+TO-PRO-3.">Pericyte mapping in cerebral slices with the far-red fluorophore TO-PRO-3.</a> Bio-protocol. 11 (22), e4222 (2021).</li><li>Mai-Morente, S. P., Marset, V. M., Blanco, F., Isasi, E. E., Abudara, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nuclear+fluorescent+dye+identifies+pericytes+at+the+neurovascular+unit.">A nuclear fluorescent dye identifies pericytes at the neurovascular unit.</a> Journal of Neurochemistry. 157 (4), 1377-1391 (2021).</li><li>Zhao, H., 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=Rationale+for+the+real-time+and+dynamic+cell+death+assays+using+propidium+iodide.">Rationale for the real-time and dynamic cell death assays using propidium iodide.</a> Cytometry. Part A: The Journal of the International Society for Analytical Cytology. 77 (4), 399-405 (2010).</li><li>Van Hooijdonk, C. A., Glade, C. P., Van Erp, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TO-PRO-3+iodide:+A+novel+HeNe+laser-excitable+DNA+stain+as+an+alternative+for+propidium+iodide+in+multiparameter+flow+cytometry.">TO-PRO-3 iodide: A novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry.</a> Cytometry. 17 (2), 185-189 (1994).</li><li>Lacar, B., Herman, P., Platel, J. C., Kubera, C., Hyder, F., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cells+regulate+capillary+blood+flow+in+the+postnatal+subventricular+zone.">Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 32 (46), 16435-16448 (2012).</li><li>Mai-Morente, S. P., Marset, V. M., Blanco, F., Isasi, E. E., Abudara, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nuclear+fluorescent+dye+identifies+pericytes+at+the+neurovascular+unit.">A nuclear fluorescent dye identifies pericytes at the neurovascular unit.</a> Journal of Neurochemistry. 157 (4), 1377-1391 (2021).</li><li>Hezel, M., Ebrahimi, F., Koch, M., Dehghani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propidium+iodide+staining:+a+new+application+in+fluorescence+microscopy+for+analysis+of+cytoarchitecture+in+adult+and+developing+rodent+brain.">Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.</a> Micron (Oxford, England). 43 (10), 1031-1038 (2012).</li><li>Mathiisen, T. M., Lehre, K. P., Danbolt, N. C., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perivascular+astroglial+sheath+provides+a+complete+covering+of+the+brain+microvessels:+An+electron+microscopic+3D+reconstruction.">The perivascular astroglial sheath provides a complete covering of the brain microvessels: An electron microscopic 3D reconstruction.</a> Glia. 58 (9), 1094-1103 (2010).</li></ol>

提交人声明，他们没有相互竞争的经济利益。

开发的是高分辨率共聚焦成像技术，用于可视化重要的脑周细胞、非重要的脑周细胞和脑切片中的微血管系统。在急性大鼠脑切片中，该过程需要用 TO-PRO-311 初始标记周细胞，然后用 IB412 标记微血管内皮细胞;随后，使用PI进行死亡周细胞的鉴定。该协议简单明了，可重复，并且高度适用于功能研究。
为了特异性地追踪神经系统内的脑周细?...

脑周细胞以其细长的突起和突出的细胞体为特征，环绕着微循环 1,2。虽然脑血流增加主要由毛细血管扩张驱动，但较小的动脉表现出较慢的扩张速度3。周细胞收缩力对毛细血管直径和周细胞形态产生影响，影响血管动力学4.脑周细胞的收缩导致毛细血管收缩，在病理情况下，过度收缩可能会阻碍红细胞流动5.多种因素，包括从蓝斑释放的去甲肾上腺素，可诱导毛细血管内的脑周细胞收缩6.周细胞在脑血流中起调节作用，表现出 20-HETE 合成，在高氧期间充当氧传感器7。氧化-硝化应激引发的脑周细胞收缩对毛细血管产生不利影响5.尽管对脑周细胞收缩进行了体内和体外研究8，但关于脑切片中活和非活脑周细胞成像的知识仍然有限。
至关重要的是，脑周细胞的组织固定后成像会影响其活力和随后的收缩性评估。此外，在神经系统疾病（例如蛛网膜下腔出血 - SAH）等情况下，脑周细胞的转基因标记无法区分活的和无活的周细胞，正如我们初步的 SAH 诱导的脑周细胞死亡研究所证实的那样9. 
为了克服这些挑战，我们使用 TO-PRO-3 标记活周细胞，同时用碘化丙啶 （PI） 染色死亡周细胞。我们使用高分辨率共聚焦成像技术来可视化脑切片中活的和无活的脑周细胞，同时在成像过程中保持切片活性。本文旨在提出一种可重复的方法，用于对脑切片中活的和无活的脑周细胞进行成像，作为探究脑周细胞对 SAH 后脑微循环影响的宝贵工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well plate</td>
			<td>ABC biochemistry</td>
			<td>ABC703006</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe</td>
			<td>Adobe Illustrator CS6 16.0.0</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>MIAOJIE</td>
			<td>225 mm x 273 mm</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Confocal imaging software</td>
			<td>Nikon</td>
			<td>NIS-Elements 4.10.00</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Nikon</td>
			<td>N-SIM/C2si</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Gas tank (5% CO2, 95% O2)</td>
			<td>PENGYIDA</td>
			<td>40L</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Glass Bottom Confocal Dishes</td>
			<td>Beyotime</td>
			<td>FCFC020-10pcs</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>EVOBOND</td>
			<td>KH-502</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Ice machine</td>
			<td>XUEKE</td>
			<td>IMS-20</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>National Institutes of Health</td>
			<td>Image J</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Inhalation anesthesia system</td>
			<td>SCIENCE</td>
			<td>QAF700</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Isolectin B 4-FITC</td>
			<td>SIGMA</td>
			<td>L2895&#8211;2MG</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447&#8211;40&#8211;7</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>7647&#8211;14&#8211;5</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10049&#8211;21&#8211;5</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>NEST Biotechnology</td>
			<td>318314</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Scientific Industries Inc</td>
			<td>Model 203</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Propidium (Iodide)</td>
			<td>Med Chem Express</td>
			<td>HY-D0815/CS-7538</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>SCIENCE</td>
			<td>QA</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard PUMP</td>
			<td>PUMP 11 ELITE Nanomite</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Thermostatic water bath</td>
			<td>OLABO</td>
			<td>HH-2</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>RT</td>
		</tr>
	</tbody>
</table>

imaging vital and non-vital brain pericytes in brain slices following subarachnoid hemorrhage

周细胞是位于大脑微循环内的关键壁细胞，在通过收缩力调节主动调节脑血流方面发挥着关键作用。传统上，它们的收缩力是通过观察特定情况下的形态变化和附近的毛细管直径变化来衡量的。然而，组织固定后、评估活力和随之而来的成像脑周周细胞的周细胞收缩力会受到影响。同样，对脑周细胞进行基因标记在区分活细胞和非活周细胞方面存在不足，特别是在蛛网膜下腔出血（SAH）等神经系统疾病中，我们的初步研究证实了脑周细胞的死亡。已经设计了一种可靠的方案来克服这些限制，能够在脑切片中同时对功能性和非功能性脑周细胞进行荧光标记。这种标记方法允许高分辨率共聚焦显微镜可视化，同时标记脑切片微血管系统。这种创新方案提供了一种评估脑周细胞收缩性、其对毛细血管直径和周细胞结构的影响的方法。在 SAH 背景下研究脑周细胞收缩力可以深入了解其对脑微循环的影响。

在正常生理条件下，脑周细胞一般不会发生细胞死亡。 图 6 说明了这种现象，黄色表示存在重要的脑周细胞;脑周细胞未显示 PI 染色，表明其活力。为了进一步研究细胞死亡后周细胞是否仍然附着在微血管系统上，在SAH大鼠模型中采用了方法，并进行了随后的成像。
已经开发了对 SAH 后脑切片中重要和非重要脑周细胞进行成像的方法。如 <strong class="xfig"...

Watch this Scientific Journal Video about Imaging Pericytes Post-Subarachnoid Hemorrhaging in Rodents at JoVE.com

作者聚焦:啮齿动物蛛网膜下腔出血后周细胞成像 

初步调查证实，蛛网膜下腔出血 （SAH） 会导致脑周细胞死亡。评估 SAH 后周细胞收缩力需要区分活的和无活的脑周细胞。因此，已经开发了一种在脑切片中同时标记活和无活脑周细胞的程序，便于使用高分辨率共聚焦显微镜进行观察。

蛛网膜下腔出血后脑切片中重要和非重要脑周细胞的成像

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility ...

我们的研究表明周细胞死亡显着增加。因此，从 SAH 后的病马开始，它证明了低生命寄生虫的数量与 SAH 后经过的时间之间存在正相关关系。在SAH和寄生虫分离技术之后，对脑切片中的重要和非重要脑寄生虫进行成像，推动了我所在领域的研究。目前，很难完全区分死去的寄生虫和死去的内皮细胞。我们的研究发现 SAH 后寄生虫的收缩依赖性细胞凋亡。我们已经开发了一种非常可靠的方案，可以同时在脑切片中对功能性和非功能性脑寄生虫进行荧光标记。

imaging-vital-non-vital-brain-pericytes-brain-slices-following

Research

JoVE Journal

Neuroscience

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

992 次观看.  The First Affiliated Hospital of Kunming Medical University. 我们的研究表明周细胞死亡显着增加。因此，从 SAH 后的病马开始，它证明了低生命寄生虫的数量与 SAH 后经过的时间之间存在正相关关系。在SAH和寄生虫分离技术之后，对脑切片中的重要和非重要脑寄生虫进行成像，推动了我所在领域的研究。目前，很难完全区分死去的寄生虫和死去的内皮细胞。我们的研究发现 SAH 后寄生虫的收缩依赖性细胞凋亡。我们已经开发了一?...

视频: 作者聚焦:啮齿动物蛛网膜下腔出血后周细胞成像 

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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) ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

脑毛细管中培养细胞细胞钙测量和钙成像研究

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...