作者想感谢苏联医学院的媒体专家, 他在视频制作、编辑和配音方面的工作。
这项工作得到了国家卫生研究院 (R01GM100474 和 R01GM072611) 的支持。

在这里和2节中描述的方法已得到佛罗里达州立大学动物保育和使用委员会 (IACUC) 的批准, 并遵循《实验室动物护理和使用指南》中规定的指南, 8th版.在这个协议中使用的所有动物都是在一个专门的实验室动物设施的房子, 直到使用。没有在体内试验是在牺牲之前进行的。动物数量是根据实验需要使用平均细胞和髓鞘收集作为指导, 以尽量减少使用。
注意:该协议描述了生成骨髓巨噬细胞 (BMDMs) (1 节), 荧光标记脑源性髓鞘碎片的制备 (2 节), 分析髓鞘碎片吞噬的一般程序 (第3节) 和髓鞘堆积物脂质积累分析的一般程序 (4 节)。在层流生物安全柜中, 应完成试剂制备、细胞采集和操作、髓鞘的收集和标记、化验性能的测定。
1. 原发骨髓巨噬细胞的生成
<ol>
	<li>全巨噬细胞培养基的制备 (CMCM) 注:骨髓巨噬细胞的生成需要使用巨噬细胞集落刺激因子 (M-CSF)。本制剂利用 L929 小鼠成纤维细胞条件培养基作为 M 型脑脊液的来源。<ol>
		<li>用50毫升高葡萄糖 (4500 毫克/l-葡萄糖) 培养细胞在145毫米菜肴中生成 L929 小鼠成纤维细胞条件培养基 (DMEM) Dulbecco 的改良鹰培养基补充5% 新出生的小牛血清和青霉素/链霉素7天。</li>
		<li>收集培养基从文化到50毫升圆锥离心管和离心机30分钟在 3500 x g, 4 ° c。</li>
		<li>过滤器清通过0.2 µm 注射器过滤器进入一个新的50毫升圆锥离心管。被适应的媒介可以被存放在-80 ° c 为6月。</li>
		<li>对高糖 DMEM, 增加5% 卷/卷的非华语, 15% 卷/卷 L929 小鼠成纤维细胞条件 DMEM, 1% 青霉素/链霉素。介质可储存在4° c 2-3 月。使用前要预热到37° c。</li>
	</ol>
	</li>
	<li>原发性骨髓细胞的采集与巨噬细胞诱导 
	注意:使用这个协议一只老鼠将产生大约2000万骨髓来源的巨噬细胞 (BMDMs)。<ol>
		<li>安乐使用标准 CO2窒息指南的8-10 周老鼠的期望数量, 其次是颈椎脱位。</li>
		<li>使用 70% (卷/卷) 乙醇对动物进行消毒: H2O, 使毛皮饱和。</li>
		<li>在腹部切开一个小开口, 并小心地拉回皮肤, 露出底层的组织。注意不要穿刺腹膜腔。</li>
		<li>从臀部开始, 在脚踝处切除两条后腿。将所收集的组织放入100毫米的培养皿中, 其中含有5-10 毫升无菌磷酸缓冲盐水 (PBS), 辅以1% 青霉素/链霉素。 
		注意:当处理几个动物时, 一定要保持在冰上的菜肴, 以限制组织退化。</li>
		<li>用手术刀从骨头上取出肌肉和结缔组织。只有股骨和胫骨用于细胞收集, 腓骨可以被丢弃。</li>
		<li>通过用手术刀将每个末端的小部分修剪, 使收集到的骨骼露出骨髓腔。</li>
		<li>使用25g 针, 冲洗骨髓腔与 CMCM 成一个50毫升圆锥离心管。一旦足够冲洗, 骨骼就会呈白色。</li>
		<li>一旦收集完成, 煽动物与18g 针30-90 秒, 产生一个单一的细胞悬浮。 
		注意:可选红细胞 (红细胞) 裂解步骤可在这一点上进行。最近有人建议, 细胞内铁含量可能影响髓鞘碎片对巨噬细胞极化的影响16。有关包含红细胞裂解步骤的信息, 请参阅 Trouplin et al。17。</li>
		<li>将悬浮液通过70µm 无菌细胞过滤器滤入新的50毫升锥形离心管中。</li>
		<li>种子收集的细胞均匀地成145毫米细胞培养皿包含15-20 毫升 CMCM。每只老鼠使用大约三培养皿。孵育文化在37° c, 5% CO2。</li>
		<li>经过72小时洗板后用无菌 PBS 去除换药细胞, 然后添加15-20mL 新鲜 CMCM。孵育文化为另外4天在37° c, 5% CO2。经过7天的总培养, 最初孤立的造血骨髓细胞将是成熟的 BMDMs (图 1)。</li>
	</ol>
	</li>
</ol>
2. 荧光标记脑源性髓鞘碎片的生成
注意:所有试剂可贮存在4° c, 可达1月。
<ol>
	<li>髓鞘碎片收集试剂的制备
	<ol>
		<li>准备 Tris·Cl 缓冲液。<ol>
			<li>到800mL 蒸馏去离子 H2o (ddiH2o) 添加 20 mL 1 米 Tris·Cl, pH 值 7.45 (20 mm 最终浓度) 和20毫升100毫米 Na2EDTA (2 毫米最终浓度)。</li>
			<li>调整 pH 值至7.45。通过0.2 µm 过滤单元, 通过 ddiH2o. 过滤器解决方案将音量调整为1000毫升。</li>
		</ol>
		</li>
		<li>准备1米蔗糖溶液。<ol>
			<li>到100毫升的 Tris·Cl 缓冲液, 加68.46 克蔗糖。用 Tris·调节音量至200毫升Cl 缓冲液。过滤器解决方案通过0.2 µm 过滤装置。</li>
		</ol>
		</li>
		<li>用 Tris·稀释 1 m 溶液, 制备200毫升的0.32 米蔗糖溶液Cl 缓冲器解决方案 0.83:0. 16 (卷/卷)。</li>
		<li>用 Tris·稀释 1 m 溶液, 制备150毫升的0.83 米蔗糖溶液Cl 缓冲器解决方案 0.83:0. 16 (卷/卷)。</li>
	</ol>
	</li>
	<li>收集脑衍生的粗髓鞘碎片 
	注意:这里描述的收集方法是为了隔离粗髓鞘碎片。<ol>
		<li>安乐10-12 只小鼠8-10 周龄使用标准 CO2窒息指南后, 颈椎脱位。</li>
		<li>解剖的大脑和放置在一个100毫米的菜, 其中含有10毫升的0.32 米蔗糖溶液。</li></ol></li></ol>把盘子放在冰上。
		<li>使用无菌手术剪刀将大脑切成大约 5 mm3大小的碎片。</li>
		<li>将组织转移到50毫升圆锥离心管上, 加入约30毫升的0.32 米蔗糖溶液。</li>
		<li>质与无菌 hand-held 旋转均质机, 直到平滑的解决方案实现。</li>
		<li>用0.32 米蔗糖溶液将匀浆的大脑稀释到90毫升的最终体积。</li>
		<li>对六38.5 毫升的薄壁聚丙烯 ultracentrifuge 管, 加入20毫升的0.83 米蔗糖溶液。</li>
		<li>轻轻地在0.83 米蔗糖溶液的顶端加入均匀化的大脑溶液, 注意不要混合两层。</li>
		<li>平衡每管与0.32 米蔗糖溶液。</li>
		<li>离心机在 10万 x g 为45分钟, 在4° c 使用适当的预 ultracentrifuge 转子。将转子的加速度和减速设置为最小值, 以减少髓鞘碎片的损失。</li>
		<li>从两个蔗糖密度的界面收集髓磷脂碎片。 
		注意: 碎片应显示为一个白色的带向中心的管。</li>
		<li>将粗髓鞘碎片组合成50毫升锥形离心管, 并使用 Tris·将体积调整到大约35毫升。Cl 缓冲液。</li>
		<li>质的粗髓鞘碎片与无菌 hand-held 旋转均质机 30-60 s。</li>
		<li>均匀地划分6干净的 ultracentrifuge 管之间的悬浮和平衡与适当的容量 Tris·Cl 缓冲液。</li>
		<li>离心机在 10万 x g 为45分钟, 在4° c 使用适当的预 ultracentrifuge 转子。将转子加速和减速设置为其最大值。</li>
		<li>由于固体白色小球现在将是可见的, 丢弃上清和 re-suspend 10-15 毫升的 Tris·颗粒Cl 缓冲液。</li>
		<li>均匀地划分2干净的 ultracentrifuge 管之间的悬浮和平衡与适当的容量 Tris·Cl 缓冲液。</li>
		<li>再次离心在 10万 x g 为45分钟在4° c 使用适当的预 ultracentrifuge 转子。将转子加速和减速设置为其最大值。</li>
		<li>丢弃上清和重5-6 毫升无菌 PBS 中的颗粒, 并在适当数量的称量1.5 毫升微离心管之间划分悬浮。</li>
		<li>离心机在 2.2万 x g 为10分钟在4° c。</li>
		<li>丢弃上清液, 确定髓鞘碎片颗粒的重量。</li>
		<li>将 PBS 中的小球再悬浮到100毫克/毫升的最后浓度。 
		注意: 十到十二的大脑足以产生10-15 毫升的100毫克/毫升髓鞘碎片。髓鞘碎片可贮存在-80 ° c 6 月。</li>
	
	
	<li>髓鞘碎片荧光标记
	<ol>
		<li>在使用前立即准备50µM 羧基琥珀酰亚胺酯 (CFSE) 溶液。<ol>
			<li>使用无菌 PBS, 稀释5毫米的 CFSE, 用100% 二甲基亚砜 (甲基亚砜) 制备的溶液, 最终工作浓度为50µM。</li>
			<li>过滤器解决方案通过0.2 µm 注射器过滤器。</li>
		</ol>
		</li>
		<li>解冻所需的100毫克/毫升髓鞘碎片和 re-suspend 与无菌29g 针。 
		注意: 在使用前, 冻结髓鞘碎片会导致它从需要悬浮的溶液中掉出来。</li>
		<li>将髓鞘碎片转移到称量1.5 毫升微离心管。</li>
		<li>离心机在 14800 x g 为10分钟在4° c。丢弃上清液。</li>
		<li>重200µL CFSE 溶液中的髓鞘碎片, 每100µL 髓鞘碎片颗粒。</li>
		<li>在室温下孵育30分钟 (RT), 免受光线的保护。</li>
		<li>离心机在 14800 x g 为10分钟在4° c。丢弃上清液。</li>
		<li>重在600-800 µL 的洗涤缓冲器 (0.2 µm 过滤器灭菌100毫米甘氨酸在 PBS) 的颗粒。</li>
		<li>离心机在 14800 x g 为10分钟在4° c。丢弃上清液。</li>
		<li>重复步骤 2.3. 8-2. 3.9 两次。</li>
		<li>最后洗涤后, 确定髓鞘碎片颗粒和 re-suspend 的重量100毫克/毫升无菌 PBS。 注: 带标记的髓鞘碎片可贮存在-80 ° c, 可长达6月。</li>
	</ol>
	</li>

3. 髓鞘碎片吞噬试验
注意:下面是观察荧光标记髓鞘碎片吞噬功能的基本方法。其他的治疗和实验条件的增加将需要由调查员优化。
<ol>
	<li>处理板的制备
	<ol>
		<li>对于每145毫米板, 收集成熟的 BMDMs 成一个15毫升圆锥离心管使用5-6 毫升的10毫米乙酸酸 (EDTA) 在 Dulbecco 的磷酸盐缓冲盐水 (dPBS) (pH 7.4)。 
		注意:不要使用胰蛋白酶, 因为它可能会改变细胞的活化状态18。</li>
		<li>在收集的细胞中加入等量的 5ml CMCM。</li>
		<li>离心机在 180 x g 为8分钟在20° c。丢弃上清液。</li>
		<li>重新挂起 CMCM 中的单元格并进行计数。</li>
		<li>对24井细胞培养板的每一个井, 在1毫升的 CMCM 中添加 1x105细胞。</li>
		<li>孵育在37° c, 5% CO2为 24 h。</li>
	</ol>
	</li>
	<li>髓鞘碎片的处理与分析
	<ol>
		<li>添加10µL 100 毫克/毫升 CFSE 标记髓鞘碎片每井 (1 毫克/毫升最终浓度)。</li>
		<li>孵育1-3 小时, 在37° c, 5% CO2。</li>
		<li>洗盘子3次, 用无菌 PBS 去除 non-engulfed 髓鞘碎片。</li>
		<li>每井加400µL 4% 甲醛。在 RT 处孵育30分钟。</li>
		<li>洗涤板2次, 无菌 PBS。</li>
		<li>每井加400µL 赫斯特33258溶液。孵育5分钟在 RT 保护免受光。</li>
		<li>用无菌 PBS 冲洗盘子2次。</li>
		<li>添加200µL 的氟凝胶与三缓冲器, 以减少漂白。</li>
		<li>使用倒置的 epi 荧光显微镜的图像细胞。 
		注意:CFSE 的激发和发射波长分别为 494 nm 和 521 nm。</li>
		<li>将 CFSE 阳性细胞数除以细胞核数, 以确定相对髓鞘碎片的摄取量。</li>
		<li>在成像之前, 保持板长达7天, 在4° c 保护免受光。</li>
	</ol>
	</li>
</ol>
4. 通过油红 O 染色定量测定胞内脂质
注意:下面是观察细胞内髓鞘-碎片衍生脂质的基本方法。由于光谱重叠, 不推荐使用 CFSE 标记的髓鞘碎片进行荧光定量。其他的治疗和实验条件的增加将需要由调查员优化。
<ol>
	<li>染色液的制备
	<ol>
		<li>准备0.5% 油红 O (破产) 染色液。<ol>
			<li>在搅拌时, 缓慢地将100毫升100% 丙二醇 (12-丙醇) 添加到0.5 克 (1-([4-Xylylazo) 二甲苯]-2-萘酚)。</li>
			<li>热溶液在95° c 为 15 min 或直到所有大微粒被溶化。 
			注意: 不要加热超过100° c。</li>
			<li>过滤器解决方案通过一块滤纸, 而仍然温暖到一个新的容器。</li>
			<li>允许解决方案在 rt 中隔夜冷却. 解决方案可在 rt 中存储长达1年。</li>
		</ol>
		</li>
		<li>准备85% 丙二醇溶液。<ol>
			<li>加入85毫升的100% 丙二醇到15毫升的 ddiH2o 搅拌至混合。解决方案可在 RT 中存储多达1年。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>处理板的制备
	<ol>
		<li>按照3节中概述的方法准备一个24井板。</li>
		<li>孵育在37° c, 5% CO2为 24 h。</li>
	</ol>
	</li>
	<li>髓鞘碎片治疗
	<ol>
		<li>每井添加10µL 100 毫克/毫升髓鞘碎片 (1 毫克/毫升最终浓度)。</li>
		<li>孵育1-3 小时, 在37° c, 5% CO2。</li>
		<li>洗盘子3次, 用无菌 PBS 去除 non-digested 髓鞘碎片。 注意: 在这一点上, 盘子可以立即进行固定或新鲜的 CMCM 可以添加, 细胞返回到孵化额外的时间点。</li>
		<li>每井加400µL 4% 甲醛。在 RT 处孵育30分钟。</li>
		<li>洗涤板2次, 无菌 PBS 然后增益染色。</li>
	</ol>
	</li>
	<li>破产法染色及分析
	<ol>
		<li>用 ddiH2O 清洗固定板3次。</li>
		<li>每井加400µL 100% 丙二醇, 孵育5分钟。 
		注意: 这将减少 ddiH2O 的结转。</li>
		<li>吸入丙二醇, 并在每口井中加入400µL 的破产解决方案。</li>
		<li>孵育8分钟, 在60° c。</li>
		<li>吸入破产解决方案。然后, 每井加400µL 85% 丙二醇, 在 RT 处孵育 5 minumintes。</li>
		<li>洗涤板3次 ddiH2O。</li>
		<li>每井加400µL 赫斯特33258溶液。在 RT 中孵育5分钟, 免受光照。</li>
		<li>用无菌 PBS 冲洗盘子2次。</li>
		<li>添加200µL 的氟凝胶与三缓冲每井。</li>
		<li>使用倒置的 epi 荧光显微镜的图像细胞。破产人可以使用标准的德州红或 DsRed 过滤器集成像。</li>
		<li>通过确定每个图像场中的破产人阳性面积并将其除以细胞核数来量化相对脂质潴留。</li>
		<li>在4° c 保护免受光照的7天内保持板材。</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Wynn, T. A., Chawla, A., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+biology+in+development,+homeostasis+and+disease.">Macrophage biology in development, homeostasis and disease.</a> Nature. 496 (7446), 445-455 (2013).</li><li>Unanue, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-presenting+function+of+the+macrophage.">Antigen-presenting function of the macrophage.</a> Annu Rev Immunol. 2, 395-428 (1984).</li><li>Cavaillon, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+macrophages.">Cytokines and macrophages.</a> Biomed Pharmacother. 48 (10), 445-453 (1994).</li><li>Ren, Y., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+the+importance+of+being+eaten.">Apoptosis: the importance of being eaten.</a> Cell Death Differ. 5 (7), 563-568 (1998).</li><li>Wang, X., 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=Macrophages+in+spinal+cord+injury:+phenotypic+and+functional+change+from+exposure+to+myelin+debris.">Macrophages in spinal cord injury: phenotypic and functional change from exposure to myelin debris.</a> Glia. 63 (4), 635-651 (2015).</li><li>Goodrum, J. F., Earnhardt, T., Goines, N., Bouldin, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+myelin+lipids+during+degeneration+and+regeneration+of+peripheral+nerve:+an+autoradiographic+study.">Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study.</a> J Neurosci. 14 (1), 357-367 (1994).</li><li>Greenhalgh, A. D., David, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+Phagocytic+Response+of+Microglia+and+Peripheral+Macrophages+after+Spinal+Cord+Injury+and+Its+Effects+on+Cell+Death.">Differences in the Phagocytic Response of Microglia and Peripheral Macrophages after Spinal Cord Injury and Its Effects on Cell Death.</a> J Neurosci. 34 (18), 6316 (2014).</li><li>Sun, X., 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=Myelin+activates+FAK/Akt/NF-kappaB+pathways+and+provokes+CR3-dependent+inflammatory+response+in+murine+system.">Myelin activates FAK/Akt/NF-kappaB pathways and provokes CR3-dependent inflammatory response in murine system.</a> PLoS One. 5 (2), e9380 (2010).</li><li>Gensel, J. C., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+its+role+in+repair+and+pathology+after+spinal+cord+injury.">Macrophage activation and its role in repair and pathology after spinal cord injury.</a> Brain Res. , 1-11 (2015).</li><li>Bourre, J. M., Pollet, S., Daudu, O., Le Saux, F., Baumann, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+consists+of+a+continuum+of+particles+of+different+density+with+varying+lipid+composition:+major+differences+are+found+between+normal+mice+and+quaking+mutants.">Myelin consists of a continuum of particles of different density with varying lipid composition: major differences are found between normal mice and quaking mutants.</a> Biochimie. 59 (10), 819-824 (1977).</li><li>Larocca, J. N., Norton, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Cell Biology. , (2001).</li><li>Norton, W. T., Poduslo, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain:+method+of+myelin+isolation.">Myelination in rat brain: method of myelin isolation.</a> J Neurochem. 21 (4), 749-757 (1973).</li><li>Wang, X. Q., Duan, X. M., Liu, L. H., Fang, Y. Q., Tan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxyfluorescein+diacetate+succinimidyl+ester+fluorescent+dye+for+cell+labeling.">Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling.</a> Acta Biochim Biophys Sin (Shanghai). 37 (6), 379-385 (2005).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77 (6), 499-508 (1999).</li><li>Deutsch, M. J., Schriever, S. C., Roscher, A. A., Ensenauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+image+analysis+approach+for+lipid+droplet+size+quantitation+of+Oil+Red+O-stained+cultured+cells.">Digital image analysis approach for lipid droplet size quantitation of Oil Red O-stained cultured cells.</a> Anal Biochem. 445, 87-89 (2014).</li><li>Kroner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+and+increased+intracellular+iron+alter+macrophage+polarization+to+a+detrimental+M1+phenotype+in+the+injured+spinal+cord.">TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord.</a> Neuron. 83 (5), 1098-1116 (2014).</li><li>Trouplin, V., 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=Marrow-derived+Macrophage+Production.">Marrow-derived Macrophage Production.</a> J Vis Exp. (81), e50966 (2013).</li><li>Chen, S., So, E. C., Strome, S. E., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Detachment+Methods+on+M2+Macrophage+Phenotype+and+Function.">Impact of Detachment Methods on M2 Macrophage Phenotype and Function.</a> J Immunol Methods. 426, 56-61 (2015).</li><li>Guo, L., 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=Rescuing+macrophage+normal+function+in+spinal+cord+injury+with+embryonic+stem+cell+conditioned+media.">Rescuing macrophage normal function in spinal cord injury with embryonic stem cell conditioned media.</a> Mol Brain. 9 (1), 48 (2016).</li><li>Bosco, D. B., 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=A+new+synthetic+matrix+metalloproteinase+inhibitor+reduces+human+mesenchymal+stem+cell+adipogenesis.">A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis.</a> PLOS ONE. 12 (2), e0172925 (2017).</li><li>Ram&#237;rez-Zacar&#237;as, J. L., Castro-Mu&#241;ozledo, F., Kuri-Harcuch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+adipose+conversion+and+triglycerides+by+staining+intracytoplasmic+lipids+with+oil+red+O.">Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O.</a> Histochem. 97 (6), 493-497 (1992).</li><li>Koopman, R., Schaart, G., Hesselink, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+oil+red+staining+permits+combination+with+immunofluorescence+and+automated+quantification+of+lipids.">Optimisation of oil red staining permits combination with immunofluorescence and automated quantification of lipids.</a> Histochem Cell Biol. 116 (1), 63-68 (2001).</li><li>Fang, 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=Lipopolysaccharide-Induced+Macrophage+Inflammatory+Response+Is+Regulated+by+SHIP.">Lipopolysaccharide-Induced Macrophage Inflammatory Response Is Regulated by SHIP.</a> J Immunol. 173 (1), (2004).</li><li>Martinez, F. O., Sica, A., Mantovani, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Front Biosci. 13, (2008).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nat Rev Immunol. 8 (12), 958-969 (2008).</li></ol>

作者没有披露。

这里描述的程序利用了新分离的原始的中枢神经系统髓鞘碎片和原发骨髓巨噬细胞。为了减少动物的开支, 我们建议在牺牲时从每只老鼠身上采集大脑和骨髓细胞。两位研究人员一起工作可以同时准备两种材料。另外, 在-80 ° c 的 PBS 中, 大脑可以在髓鞘碎片隔离之前用抗生素补充。我们的经验是, 大脑可以在这些条件下维持1月, 而不会有明显的髓鞘碎片生成潜能的损失。
CFSE ?...

骨髓源性巨噬细胞 (BMDMs) 是先天和适应性免疫系统之间的重要联系。作为抗原呈递细胞 (apc), 他们可以通过抗原呈递和细胞因子释放1,2,3与淋巴细胞进行通信。然而, 作为专业吞噬, 他们的主要功能是清除病原体, 衰老细胞和细胞碎片1,4。继脊髓损伤 (SCI), 大量髓鞘碎片是由死亡的突, 细胞类型负责中枢神经系统轴突鞘5。我们和其他人已经表明, 清除髓鞘碎片主要是渗透 BMDMs 的责任5,6,7。然而, 在脊髓损伤部位吞没髓鞘碎片被建议将这些正常的抗炎细胞转变为炎状态5,8,9。BMDMs 是脊髓损伤中神经炎症的重要介质, 是临床的主要靶点。
为了帮助研究 BMDMs 在损伤脊髓中的影响, 我们开发了一个体外模型, 直接探讨 BMDMs 对髓鞘碎片的反应。为了提高生物学相关性, 在这些调查中使用了小鼠 BMDMs 和新近分离的髓鞘碎片。因此, 这里介绍的方法也详述了原小鼠 BMDMs 的分离和培养, 以及用于分离鼠中枢神经系统衍生髓鞘碎片的改良蔗糖梯度技术10,11,12。髓鞘碎片可以很容易地标记为荧光染料, 羧基琥珀酰亚胺酯 (CFSE), 以跟踪其内部化的 BMDMs. CFSE 是非常适合这个应用, 因为它是 non-cytotoxic, 其狭窄的荧光光谱允许多路传输与其他荧光探针13,14。继吞噬后, 髓鞘碎片脂通过溶酶体转运, 并以中性脂质包装成细胞内脂滴5。为了量化这种细胞内脂质的积累, 我们提出了一个油红 O (破产) 染色方法优化定量图像分析。这个简单的染色方法产生可靠的重现性结果和量化的15。这些方法有助于研究的髓鞘碎片吞噬和脂质保留与有限的专用设备。

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">DMEM</td>
			<td style="width:149px;">GE Healthcare Life Sciences</td>
			<td style="width:115px;">SH30243.01</td>
			<td style="width:225px;">High glucose with L-glutamine, sodium pyruvate</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:180px;">Penicillin-Streptomycin Solution</td>
			<td style="width:149px;">Corning</td>
			<td style="width:115px;">30-002-CI</td>
			<td style="width:225px;">100X Solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">New Born Calf Serum (NCS)</td>
			<td style="width:149px;">Rocky Mountain Biologics</td>
			<td style="width:115px;">NBS-BBT-5XM</td>
			<td style="width:225px;">United States Origin</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:180px;">NCTC clone 929 [L cell, L-929, derivative of Strain L]&#160;</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:115px;">CCL-1</td>
			<td style="width:225px;">L929 Cell Line of Conditioned Media Preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">24-well Cell Culture Plates</td>
			<td style="width:149px;">VWR</td>
			<td style="width:115px;">10062-896&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:180px;">Cell Culture Dish</td>
			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:115px;">639960</td>
			<td style="width:225px;">Polystyrine, 145/20mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">CFSE Cell Proliferation Kit</td>
			<td style="width:149px;">Thermo Fisher</td>
			<td style="width:115px;">C34570</td>
			<td style="width:225px;">DMSO for Reconsitution Provided</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Fluoro-gel with Tris Buffer&#160;</td>
			<td style="width:149px;">Electron Microscopy Sciences</td>
			<td style="width:115px;">17985-11</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:180px;">Oil Red O</td>
			<td style="width:149px;">Sigma Aldrich</td>
			<td style="width:115px;">O0625</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:180px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Materials</td>
			<td style="width:149px;">Company</td>
			<td style="width:115px;">Catalog Number</td>
			<td style="width:225px;">Comments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Ultracentrifuge Tubes</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:115px;">326823</td>
			<td style="width:225px;">Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:180px;">SW 32 Ti Ultracentrifuge Rotor</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:115px;">369650</td>
			<td style="width:225px;">SW 32 Ti Rotor, Swinging Bucket, Titanium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Hand Held Rotary Homogenizer</td>
			<td style="width:149px;">Fisher Science</td>
			<td style="width:115px;">08-451-71</td>
			<td style="width:225px;"></td>
		</tr>
	</tbody>
</table>

in vitro phagocytosis of myelin debris by bone marrow-derived macrophages

骨髓源性巨噬细胞 (BMDMs) 是成熟的白细胞, 具有重要的生理作用, 作为专业吞噬能够清除各种微粒。正常情况下, BMDMs 受中枢神经系统 (CNS) 的限制, 但在受伤后, 他们可以很容易地渗入。一旦在受损的中枢神经组织内, BMDMs 是主要的细胞类型, 负责清除损伤衍生的细胞碎片, 包括大量的富含脂质的髓鞘碎片。中枢神经系统内 BMDM 浸润和髓鞘碎片吞噬的病理影响是复杂的, 不太清楚。这里描述的协议, 允许直接的体外研究 BMDMs 在中枢神经系统损伤的情况下。我们覆盖小鼠 BMDM 分离和培养, 髓鞘碎片制备, 并测定 BMDM 髓鞘碎片吞噬功能。这些技术在不需要重要的专用设备或材料的情况下产生可靠的可量化结果, 但可以很容易地定制以满足研究人员的需要。

BMDMs 的 CFSE 标记髓鞘碎片的治疗应产生清晰的内部化 (图 2)。虽然3小时的互动时间是足够的 BMDMs, 以吞噬足够的补充髓鞘碎片, 为强健的下游检测, 细胞内积累可以观察到, 只需1小时的互动。然而, 一些髓鞘碎片可能仍然存在于细胞表面洗涤后。这可能是由于洗涤不足, 或在吞噬的早期阶段没有完全内化的微粒。
虽然 BMDMs 可以迅速地将髓鞘碎片化, 但在破?...

Watch this Scientific Journal Video about In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages at JoVE.com

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

本文采用荧光标记髓鞘碎片和胞内脂滴染色的方法, 对小鼠骨髓源性巨噬细胞的吞噬能力进行评估。

体外骨髓源性巨噬细胞对髓鞘碎片的吞噬

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

in-vitro-phagocytosis-myelin-debris-bone-marrow-derived

Research

JoVE Journal

Neuroscience

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

12.6K 次观看.  Florida State University. 本文采用荧光标记髓鞘碎片和胞内脂滴染色的方法, 对小鼠骨髓源性巨噬细胞的吞噬能力进行评估。

视频: In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

体外刺激对龟脑神经的运动测量

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

小鼠原代多元体的体外模拟？

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance imaging (fMRI) studies indicates that the anterior cingulate (ACC) and the lateral prefrontal cortices (latPFC) are essential nodes subserving cognitive control. However, because of its limited temporal resolution, fMRI cannot accurately reflect the timing and nature of their presumed interplay. The present study combines distributed source modeling of the temporally precise magnetoencephalography (MEG) signal with structural MRI in the form of &#34;brain movies&#34; to: (1) estimate the cortical areas involved in cognitive control (&#34;where&#34;), (2) characterize their temporal sequence (&#34;when&#34;), and (3) quantify the oscillatory dynamics of their neural interactions in real time. Stroop interference was associated with greater event-related theta (4 - 7 Hz) power in the ACC during conflict detection followed by sustained sensitivity to cognitive demands in the ACC and latPFC during integration and response preparation. A phase-locking analysis revealed co-oscillatory interactions between these areas indicating their increased neural synchrony in theta band during conflict-inducing incongruous trials. These results confirm that theta oscillations are fundamental to long-range synchronization needed for integrating top-down influences during cognitive control. MEG reflects neural activity directly, which makes it suitable for pharmacological manipulations in contrast to fMRI that is sensitive to vasoactive confounds. In the present study, healthy social drinkers were given a moderate alcohol dose and placebo in a within-subject design. Acute intoxication attenuated theta power to Stroop conflict and dysregulated co-oscillations between the ACC and latPFC, confirming that alcohol is detrimental to neural synchrony subserving cognitive control. It interferes with goal-directed behavior that may result in deficient self-control, contributing to compulsive drinking. In sum, this method can provide insight into real-time interactions during cognitive processing and can characterize the selective sensitivity to pharmacological challenge across relevant neural networks.

This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication.

酒精中毒在认知控制过程中对前小管神经同步的破坏

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

气相臭受体活化的实时体外监测

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

多发性硬化症的莱索莱西汀鼠模型中用于测量骨髓含量的 体内 发射断层扫描成像

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

体外骨髓源性巨噬细胞对髓鞘碎片的吞噬

摘要

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此视频中的章节

小鼠视网膜神经节细胞的转染在体内电穿孔

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

同时

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

体外刺激对龟脑神经的运动测量

小鼠原代多元体的体外模拟？

酒精中毒在认知控制过程中对前小管神经同步的破坏

气相臭受体活化的实时体外监测

多尾根结鱼巨噬细胞的快速隔离

多发性硬化症的莱索莱西汀鼠模型中用于测量骨髓含量的体内发射断层扫描成像

体外骨髓源性巨噬细胞对髓鞘碎片的吞噬

摘要

探索更多视频

此视频中的章节

小鼠视网膜神经节细胞的转染在体内电穿孔

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

同时

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

体外刺激对龟脑神经的运动测量

小鼠原代多元体的体外模拟？

酒精中毒在认知控制过程中对前小管神经同步的破坏

气相臭受体活化的实时体外监测

多尾根结鱼巨噬细胞的快速隔离

多发性硬化症的莱索莱西汀鼠模型中用于测量骨髓含量的 体内 发射断层扫描成像

多发性硬化症的莱索莱西汀鼠模型中用于测量骨髓含量的体内发射断层扫描成像