Name: a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels
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这项工作得到了国家卫生研究院 (R01-NS084941) 和亚利桑那生物医学研究委员会 (ADHS16-162406) 提供给 PTR 的赠款的支持。T32-HL007249 已经获得了从博士后任命到国家卫生培训补助金研究所的支持。

下面概述的所有程序都已由一个机构动物护理和使用委员会 (IACUC) 批准, 并符合国家卫生研究院和动物研究: 报告体内实验 (到达) 指南。协议的过程流程在图 1中描述。
1. 程序的设置
<ol><li>准备脑微血管缓冲 (BMB)。从称重54.66 克 d-甘露醇, 1.90 克 EGTA, 1.46 克 2-氨基-2-(羟甲基)-13-丙二醇 (即, 三) 基为清洁烧杯开始。加1.0 升去离子水。使用磁力搅拌器混合 BMB 缓冲器的元件。一旦溶液彻底混合, 将 pH 值调整为 7.4, 使用1.0 米 HCl。</li>
	<li>在麻醉动物之前, 准备26% 葡聚糖 (兆瓦 7.5万) 溶液 (w/v)。按照以下步骤中所述, 准备葡聚糖溶液。 注意: 提前准备这个解决方案是至关重要的, 因为葡聚糖可以大约 1.5-2 小时的时间溶解在水中的缓冲液中。<ol>
<li>将粉葡聚糖 (26 克/100 毫升缓冲) 称量成干净的烧杯。</li>
		<li>测量出适当的 BMB 体积, 并分配到一个单独的, 干净的烧杯。</li>
		<li>慢慢地将 BMB 倒入含粉状葡聚糖的烧杯中。用玻璃搅拌棒在 BMB 的浇注过程中, 手工混合粉葡聚糖。</li>
		<li>使用前立即将 pH 值调整为7.4 和1.0 米 HCl。 注: 典型的从12个大大鼠 (男性或女性) 脑微血管的分离; 3 月的年龄; 200-250 克每一个) 需要200毫升葡聚糖溶液 (即52毫升的 BMB)。</li>
	</ol>
</li>
</ol>2. 大大鼠脑组织的提取
<ol><li>经过所需的实验治疗, 麻醉大大鼠使用氯胺酮 (50 毫克/千克; 2.5 毫升/千克 ip) 和甲苯噻嗪 (10 毫克/毫升; 2.5 毫升/千克 i, p.)。稀释氯胺酮和甲苯噻嗪在0.9% 盐水到最后浓度的20毫克/毫升和4.0 毫克/毫升分别。弄死动物通过斩首使用削尖的断头台根据 IACUC 指南。对雄性和雌性大大鼠使用相同的手术方法。</li>
	<li>用手术剪刀做一个横切口切除大鼠颅骨的皮肤。</li>
	<li>使用 rongeurs, 小心地取出颅骨板并暴露大脑。</li>
	<li>用刮刀取出大脑。将大脑分离, 将孤立的脑组织放置在含有5毫升 BMB 的50毫升圆锥管中。添加 1.0 ul 的蛋白酶抑制剂鸡尾酒每1.0 毫升的 BMB 缓冲区立即使用前。</li>
</ol>3. 脑处理
<ol><li>将脑组织从50毫升锥形管转移到干净的培养皿中。</li>
	<li>使用镊子, 轻轻滚动12.5 厘米直径滤纸的大脑, 以消除外层脑膜, 这是松散地坚持到大脑皮层。轻轻地按下脑组织对滤纸和卷的组织再次。在此步骤中经常打开过滤器纸张。</li>
	<li>用镊子将脉络丛从大脑半球分离出来。 注: 脉络丛出现于脑室表面的透明膜组织。</li>
	<li>用镊子轻轻地压扁脑组织, 取出残余的脑膜和嗅觉灯泡。</li>
	<li>将皮质脑组织放入冷冻玻璃砂浆中。加入5.0 毫升的 BMB 含有蛋白酶抑制剂鸡尾酒的砂浆。</li>
	<li>使用架空功率均质机, 融汇脑组织使用15上下中风在 3700 rpm。使用10毫升砂浆和杵组织磨床进行均匀化。在每个样品的均匀化之间, 用70% 乙醇清洗杵。 注意: 均匀性笔画应在速度和幅度上保持一致。</li>
	<li>将匀浆倒入标签离心管。</li>
</ol>4. 离心步骤
<ol><li>添加8.0 毫升26% 葡聚糖溶液, 每个标签离心管包含脑匀浆。</li>
	<li>反转两次管, 然后彻底涡流样品。使用多个角度进行涡流, 以确保大脑匀浆液与26% 葡聚糖溶液彻底混合。</li>
	<li>离心机样品在 5000 x g为15分钟在4°c。 注意: 对于某些分析, 将脑微血管与脑实质进行比较是有益的。如果需要对脑实质中蛋白质表达的评估, 从这个离心步骤 (即脑实质分数) 中的上清可以收集和储存在-80 摄氏度, 以供将来使用。</li>
	<li>使用真空吸尘器和玻璃巴斯德吸管去除上清液。 注意: 必须注意不要干扰颗粒。否则, 将减少所采集的脑微血管数量, 这将显著降低该过程中蛋白质的产量。</li>
	<li>并用重悬在含有蛋白酶抑制剂鸡尾酒的5.0 毫升 BMB 中的颗粒 (即 1.0 ul 蛋白酶抑制剂鸡尾酒每1.0 毫升的 BMB 缓冲器)。涡流颗粒, 确保彻底搅拌。</li>
	<li>将8.0 毫升26% 葡聚糖添加到每个离心管和涡流中, 如本协议步骤 4.1-4.2 所述。</li>
	<li>离心机样品在 5000 x g为15分钟在4°c。</li>
	<li>使用真空瓶和玻璃吸管, 吸入上清, 并确保含有脑微血管的小球不会被破坏。 注: 附着在管壁上的多余材料不含微血管, 应仔细清洗和清除。</li>
	<li>重复步骤4.5 至 4.8, 另外两次。</li>
	<li>4葡聚糖离心步骤完成后, 在每个颗粒和涡流中加入5.0 毫升的 BMB, 以并用重悬样品。 注意: 在步骤4.10 完成后, 可以收集整个微血管来分析蛋白质的定位。这可以通过采取 50 ul 整除重新悬浮微血管颗粒和涂抹在玻璃显微镜幻灯片上完成。微血管然后热固定在95°c 10 分钟在一个加热块, 然后在冰冷的乙醇固定, 另外10分钟, 然后可以存储在4°c, 直到需要进行成像研究。</li>
</ol>5. 离心制备全脑微血管膜标本
<ol><li>将样品转移到冷冻玻璃均质机上。</li>
	<li>使用架空功率均质机, 融汇脑组织使用8-10 上下中风在 3000 rpm。采用10毫升砂浆和杵组织磨床进行均匀化。在每样样品均匀化后用70% 乙醇清洗杵。</li>
	<li>将样品转移到清洁 ultracentrifuge 管。数和重量每个单独管。平衡和对管之前加载到 ultracentrifuge 转子。 注: 所有 ultracentrifuge 管的称量和配对必须与瓶盖一起进行, 以确保准确测量管的重量。</li>
	<li>离心机样品在 15万 x g为1小时在4°c。</li>
	<li>使用真空瓶和玻璃巴斯德吸管, 吸入上清, 小心不要扰乱毛细血管膜颗粒。</li>
	<li>根据颗粒的大小, 添加适当的存储缓冲量, 这是由去离子水和 BMB 的 1:1 (v/v) 比率组成。通常, 颗粒在 400-500 ul 的存储缓冲区中悬浮。添加蛋白酶抑制剂鸡尾酒到存储缓冲区的比率为 1.0 ul 每1.0 毫升的存储缓冲区。</li>
	<li>在存储缓冲区中并用重悬颗粒的涡流样品。</li>
	<li>使用玻璃巴斯德吸管, 转移毛细管膜样品到一个标记的1.5 毫升离心管。</li>
	<li>通过针头注射器 5x, 以确保样品完全混合, 并没有细胞聚集存在。</li>
	<li>将样品存放在摄氏-80 摄氏度, 直到需要进行分析。 注: 用布拉德福德法测定各微血管膜样品的蛋白质含量。</li>
</ol>

<ol>
	<li>Rolfe, D. F., Brown, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+energy+utilization+and+molecular+origin+of+standard+metabolic+rate+in+mammals.">Cellular energy utilization and molecular origin of standard metabolic rate in mammals.</a> Physiol Rev. 77 (3), 731-758 (1997).</li><li>Brzica, H., Abdullahi, W., Ibbotson, K., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Transporters+in+Central+Nervous+System+Drug+Delivery+and+Blood-Brain+Barrier+Protection:+Relevance+to+Treatment+of+Stroke.">Role of Transporters in Central Nervous System Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke.</a> J Cent Nerv Syst Dis. 9, 1179573517693802 (2017).</li><li>Ronaldson, P. T., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+transporters:+promoting+blood-brain+barrier+repair+in+response+to+oxidative+stress+injury.">Targeting transporters: promoting blood-brain barrier repair in response to oxidative stress injury.</a> Brain Res. 1623, 39-52 (2015).</li><li>Witt, K. A., Mark, K. S., Hom, S., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hypoxia-reoxygenation+on+rat+blood-brain+barrier+permeability+and+tight+junctional+protein+expression.">Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression.</a> Am J Physiol Heart Circ Physiol. 285 (6), H2820-H2831 (2003).</li><li>McCaffrey, G., 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=Occludin+oligomeric+assemblies+at+tight+junctions+of+the+blood-brain+barrier+are+altered+by+hypoxia+and+reoxygenation+stress.">Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress.</a> J Neurochem. 110 (1), 58-71 (2009).</li><li>Lochhead, J. J., 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=Oxidative+stress+increases+blood-brain+barrier+permeability+and+induces+alterations+in+occludin+during+hypoxia-reoxygenation.">Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation.</a> J Cereb Blood Flow Metab. 30 (9), 1625-1636 (2010).</li><li>Lucke-Wold, B. 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=Bryostatin-1+Restores+Blood+Brain+Barrier+Integrity+following+Blast-Induced+Traumatic+Brain+Injury.">Bryostatin-1 Restores Blood Brain Barrier Integrity following Blast-Induced Traumatic Brain Injury.</a> Mol Neurobiol. 52 (3), 1119-1134 (2015).</li><li>Campos, C. R., Ocheltree, S. M., Hom, S., Egleton, R. D., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+inhibition+prevents+inflammatory+pain+induced+changes+in+the+blood-brain+barrier.">Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier.</a> Brain Res. , 6-13 (2008).</li><li>Ronaldson, P. T., Demarco, K. M., Sanchez-Covarrubias, L., Solinsky, C. M., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta+signaling+alters+substrate+permeability+and+tight+junction+protein+expression+at+the+blood-brain+barrier+during+inflammatory+pain.">Transforming growth factor-beta signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain.</a> J Cereb Blood Flow Metab. 29 (6), 1084-1098 (2009).</li><li>Seelbach, M. J., Brooks, T. A., Egleton, R. D., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+inflammatory+hyperalgesia+modulates+morphine+delivery+to+the+brain:+a+role+for+P-glycoprotein.">Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein.</a> J Neurochem. 102 (5), 1677-1690 (2007).</li><li>Ronaldson, P. T., Finch, J. D., Demarco, K. M., Quigley, C. E., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+pain+signals+an+increase+in+functional+expression+of+organic+anion+transporting+polypeptide+1a4+at+the+blood-brain+barrier.">Inflammatory pain signals an increase in functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier.</a> J Pharmacol Exp Ther. 336 (3), 827-839 (2011).</li><li>Pop, 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=Early+brain+injury+alters+the+blood-brain+barrier+phenotype+in+parallel+with+beta-amyloid+and+cognitive+changes+in+adulthood.">Early brain injury alters the blood-brain barrier phenotype in parallel with beta-amyloid and cognitive changes in adulthood.</a> J Cereb Blood Flow Metab. 33 (2), 205-214 (2013).</li><li>Thompson, B. J., 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=Hypoxia/reoxygenation+stress+signals+an+increase+in+organic+anion+transporting+polypeptide+1a4+(Oatp1a4)+at+the+blood-brain+barrier:+relevance+to+CNS+drug+delivery.">Hypoxia/reoxygenation stress signals an increase in organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier: relevance to CNS drug delivery.</a> J Cereb Blood Flow Metab. 34 (4), 699-707 (2014).</li><li>Tome, M. E., 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=P-glycoprotein+traffics+from+the+nucleus+to+the+plasma+membrane+in+rat+brain+endothelium+during+inflammatory+pain.">P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain.</a> J Cereb Blood Flow Metab. 36 (11), 1913-1928 (2016).</li><li>Slosky, L. M., 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=Acetaminophen+modulates+P-glycoprotein+functional+expression+at+the+blood-brain+barrier+by+a+constitutive+androstane+receptor-dependent+mechanism.">Acetaminophen modulates P-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism.</a> Mol Pharmacol. 84 (5), 774-786 (2013).</li><li>Artus, C., 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=The+Wnt/planar+cell+polarity+signaling+pathway+contributes+to+the+integrity+of+tight+junctions+in+brain+endothelial+cells.">The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells.</a> J Cereb Blood Flow Metab. 34 (3), 433-440 (2014).</li><li>Yu, 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=Long-term+exposure+to+ethanol+downregulates+tight+junction+proteins+through+the+protein+kinase+Calpha+signaling+pathway+in+human+cerebral+microvascular+endothelial+cells.">Long-term exposure to ethanol downregulates tight junction proteins through the protein kinase Calpha signaling pathway in human cerebral microvascular endothelial cells.</a> Exp Ther Med. 14 (5), 4789-4796 (2017).</li><li>Abdullahi, W., Brzica, H., Ibbotson, K., Davis, T. P., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-9+increases+the+functional+expression+of+organic+anion+transporting+polypeptide+1a4+at+the+blood-brain+barrier+via+the+activin+receptor-like+kinase-1+receptor.">Bone morphogenetic protein-9 increases the functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier via the activin receptor-like kinase-1 receptor.</a> J Cereb Blood Flow Metab. 37 (7), 2340-2345 (2017).</li><li>Mesev, E. V., Miller, D. S., Cannon, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ceramide+1-Phosphate+Increases+P-Glycoprotein+Transport+Activity+at+the+Blood-Brain+Barrier+via+Prostaglandin+E2+Signaling.">Ceramide 1-Phosphate Increases P-Glycoprotein Transport Activity at the Blood-Brain Barrier via Prostaglandin E2 Signaling.</a> Mol Pharmacol. 91 (4), 373-382 (2017).</li><li>Betz, A. L., Csejtey, J., Goldstein, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hexose+transport+and+phosphorylation+by+capillaries+isolated+from+rat+brain.">Hexose transport and phosphorylation by capillaries isolated from rat brain.</a> Am J Physiol. 236 (1), C96-C102 (1979).</li><li>Yousif, S., Marie-Claire, C., Roux, F., Scherrmann, J. M., Decleves, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+drug+transporters+at+the+blood-brain+barrier+using+an+optimized+isolated+rat+brain+microvessel+strategy.">Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy.</a> Brain Res. 1134 (1), 1-11 (2007).</li><li>McCaffrey, G., 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=Tight+junctions+contain+oligomeric+protein+assembly+critical+for+maintaining+blood-brain+barrier+integrity+in+vivo.">Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo.</a> J Neurochem. 103 (6), 2540-2555 (2007).</li><li>Brzica, 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=The+liver+and+kidney+expression+of+sulfate+anion+transporter+sat-1+in+rats+exhibits+male-dominant+gender+differences.">The liver and kidney expression of sulfate anion transporter sat-1 in rats exhibits male-dominant gender differences.</a> Pflugers Arch. 457 (6), 1381-1392 (2009).</li><li>Ronaldson, P. T., Bendayan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+viral+envelope+glycoprotein+gp120+produces+oxidative+stress+and+regulates+the+functional+expression+of+multidrug+resistance+protein-1+(Mrp1)+in+glial+cells.">HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells.</a> J Neurochem. 106 (3), 1298-1313 (2008).</li><li>Pustylnikov, S., Sagar, D., Jain, P., Khan, Z. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+C-type+lectins-mediated+host-pathogen+interactions+with+dextran.">Targeting the C-type lectins-mediated host-pathogen interactions with dextran.</a> J Pharm Pharm Sci. 17 (3), 371-392 (2014).</li><li>Obermeier, B., Daneman, R., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+maintenance+and+disruption+of+the+blood-brain+barrier.">Development, maintenance and disruption of the blood-brain barrier.</a> Nat Med. 19 (12), 1584-1596 (2013).</li><li>Abdullahi, W., Davis, T. P., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Expression+of+P-glycoprotein+and+Organic+Anion+Transporting+Polypeptides+at+the+Blood-Brain+Barrier:+Understanding+Transport+Mechanisms+for+Improved+CNS+Drug+Delivery?.">Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?.</a> AAPS J. 19 (4), 931-939 (2017).</li></ol>

作者没有什么可透露的。

本文介绍了一种简单有效的从大鼠脑组织中分离的微血管制备膜蛋白样品的方法。在文献13、20、21、22中, 已报告了几种分离大鼠脑微血管和/或生成来自孤立微孔的膜制剂的方法。,24. 虽然上文所述的微血管隔离协议在原理上是相似的, 但这一方法已经得到了优化, 能够制备出具有优良蛋白质产量的单一实验动物的膜样品。这一进展消除了需要从至少三大大鼠的微血管, 以获得满意的蛋白质富集样本, 用于各种蛋白质分析方法。这些方法包括, 但不限于, 西方污点分析, 斑点印迹, 和共同免疫沉淀。应该指出的是, 从多个实验动物中聚集微血管以生成单个样本的做法大大减少了群体间的变异性。在这个程度上, 微血管的汇集可能导致数据与减少的传播不代表真正的变异在被观察的蛋白质表达在实验动物的人口。由于可以从单一实验动物中制备样本, 因此捕获了研究种群的真实变异性, 这使得实验数据更能代表病理生理学或药理过程。考试。另一个好处是, 微血管膜样品可以用减少的实验动物数量来制备。
在执行本议定书时, 最关键的考虑可能包括离心步骤。事实上, 由于其年龄、制造商和/或整体状况和完整性, 离心机在实验室之间可能会有所不同。这可能导致实际的g级别 (或 rpm 级别) 从旋转转到旋转的显著变化。这种变化在样本完整性中的作用可以通过在同一转子中的单个比较分析中的所有样品的离心来最小化。例如, 如果一个转子只有十二个可供取样管的插槽, 那么单个实验中的微血管样本的分离和制备将限于十二。这个考虑确保准备好的样品具有相当的质量, 并且实验组间平均蛋白表达的任何变异性都可以分配给治疗条件本身。
另外一个考虑因素包括准备26% 葡聚糖溶液, 这是需要通过差异离心分离脑微血管和脑实质组织。葡聚糖是一种葡萄糖聚合物, 其发病率为α16联的单位, 通常具有线性结构25。它还具有较高的水结合能力。例如, 1 克葡聚糖 75 (即, 在本议定书中使用的平均分子量为 7.5万 Da 的葡聚糖) 保留约20-25 毫升的水。葡聚糖的这种性质可以在溶解粉末到水中的缓冲中带来深刻的挑战。通过加热葡聚糖溶液达到40摄氏度的最高温度, 可以改善水的溶解度。在加热时, 聚合物与水分子相互作用较少, 从而导致葡聚糖在溶液中采用较不膨胀的构象 (即较低的保水度)。加热到更高的温度将导致葡聚糖聚合物分解, 因此, 它将无效作为分离试剂。此外, 在使用前, 右旋糖酐溶液的 pH 值必须立即调整为7.4。26% 葡聚糖的溶液可以经历显著的 pH 变化, 甚至在准备后1-2 小时。由于这些考虑, 26% 葡聚糖溶液必须在实验当天准备好, 但在分离脑组织之前有足够的时间, 以允许溶解和适当调整 pH 值。
该协议的一个局限性是在微血管制剂中存在其他细胞类型的神经血管单元 (NVU)。除内皮细胞外, NVU 由各种细胞成分组成, 包括胶质细胞 (如星形细胞、小胶质)、毛细血管和神经元3、26、27。鼠脑微血管的分离典型表现为血管内皮细胞的富集;然而, 神经标记蛋白的表达以及胶质纤维酸性蛋白 (GFAP) 的表达也得到了证实。此外, 由于与血管内皮的亲密联系, 毛细血管在样品制备中存在。目前, 几乎不可能将脑微血管与实验动物分离, 并消除所有毛细血管、神经元和星形胶质细胞。然而, 该协议已通过多个离心步骤, 使样品丰富的内皮细胞 (即, 如通过改善表达 PECAM-1 后, 每个离心步骤) 与有限的存在其他细胞类型 (例如, 在每个离心步骤之后, 神经元标记蛋白 synaptophysin-1 表达的减少表示。
总的来说, 这种隔离大鼠脑微血管和制备膜样品的方法可以在任何实验室中适应, 在病理生理学或药理学条件下, 有兴趣研究 BBB 蛋白。例如, 本协议提供微血管样本, 可用于研究在 BBB218内在表达的药物转运蛋白。这种方法的健壮性使观察 Oatp1a4 的离散变化, 以响应药理刺激, 实验数据, 已通知设计严谨的研究, 以评估转运功能和调节在脑屏障。上述协议也可以应用于蛋白质分析的紧密结蛋白和 adherens 结蛋白, 努力了解血脑屏障在生理条件下, 并研究脑屏障完整性的变化, 以应对病理生理学和药理压力。显然, 这个协议可以适应在脑屏障领域的多个应用, 因此, 它代表了一个简单而健壮和可重复的方法, 可以纳入脑屏障研究需要蛋白质分析。

血脑屏障 (BBB) 存在于中枢神经系统 (CNS) 与系统循环之间的界面, 在维持脑稳态中起着至关重要的作用。具体地说, 脑屏障功能精确控制大脑胞外液中的溶质浓度, 并有效地提供脑组织所需的养分, 以满足中枢神经系统1的相当大的新陈代谢需求。这些作用意味着脑屏障, 主要存在于微血管内皮细胞的水平, 必须拥有离散机制, 使某些物质能够进入大脑实质, 同时确保潜在的有害外来化合物不能积累。事实上, 脑微血管内皮细胞不是 fenestrated 和表现有限的吞饮, 这确保缺乏非选择性通透性 2.此外, 脑微血管内皮细胞表达紧密连接和 adherens 结蛋白, 在相邻的内皮细胞之间形成物理 "海豹", 并极大地限制血液传播物质在脑中的旁细胞扩散。实质。事实上, 内源和外源物质的选择性渗透性需要吸收和流出转运体的功能表达式3。总的来说, 紧密的连接, adherens 连接, 和运输工作, 以保持独特的屏障性能的血屏障。
脑屏障是一个动态屏障, 反应生理, 病理生理学和药理刺激。例如, 缺氧/复氧应力已被证明可以调节临界紧致结蛋白 (即occludin、zonulae occluden-1 (ZO-1)) 的表达, 这与增加旁细胞通透性的血管标记有关, 如作为蔗糖4,5,6。在创伤性脑损伤的设置方面也有类似的观察:7和外围炎症疼痛8,9。这些同样的疾病也可以调节在 BBB10,11,12,13,14的传输机制。事实上, 缺氧/复氧损伤增强了有机阴离子转运多肽 1a4 (Oatp1a4) 在脑屏障中的功能表达, 这可能导致特定 Oatp 运输基质的血液到大脑传输显著增加, 如胆酸和阿托伐他汀13。脑屏障的特性也可以通过药物治疗本身来改变, 这种机制可以为大脑和药物相互作用的药物有效性的深刻变化奠定基础。例如, 扑热息痛靶向脑微血管内皮细胞核受体信号机制, 增加临界外排转运蛋白 p-糖蛋白 (p gp) 的功能表达, 并修改时间依赖性镇痛由吗啡、阿片类镇痛药和建立的 P gp 运输基质15授予。彻底了解血脑屏障的变化, 这可能是由疾病或药物引起的, 也需要识别和鉴定特定的调控机制, 控制这些修改。事实上, 在控制紧结蛋白的分子表达的脑微血管内皮细胞中发现了离散信号通路16,17和运输商15, 18,19。这些观察结果表明, 复杂的分子通路参与了对脑屏障紧密连接和转运体的健康和疾病的调控。
在研究脑屏障的一个重大挑战是一个简单有效的方法, 从实验动物的大脑组织中分离微血管和后续制备膜样品的绝对要求。这些样品必须准备好, 使它们既丰富的脑微血管内皮细胞和有限的存在其他细胞类型。在过去的几年中, 科学家在科学文献13、20、21、22中报告了多种从啮齿动物脑中分离微血管的方法。本文介绍了一种简单、健壮、可重现的方法, 用于分离大鼠脑中的微血管, 并制备出一种能用于蛋白质表达分析的内皮膜富集样品。这种微血管隔离协议的优点是能够获得高质量的样品制备, 并能从单个实验动物那里得到足够的蛋白质产量。这样就可以考虑蛋白质表达中动物间的变异。此项协议的进展大大提高了脑屏障研究的健壮性, 因为现在可以避免对脑屏障蛋白变化的真实程度进行过度估计 (或估计不足)。另外, 加入葡聚糖的多重离心步骤, 可以改善实验样品中微血管的富集, 同时有助于去除不必要的细胞成分, 如神经元。

<table><tbody><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>#P8340</td><td>Component of brain microvessel buffer</td></tr><tr><td>D-mannitol</td><td>Sigma-Aldrich</td><td>#M4125</td><td>Component of brain microvessel buffer</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>#E3889</td><td>Component of brain microvessel buffer</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>#T1503</td><td>Component of brain microvessel buffer</td></tr><tr><td>Dextran (MW 75,000)</td><td>Spectrum Chemical Mftg Corp</td><td>#DE125</td><td>Dextran used in centrifugation steps to separate microvessels from brain parenchyma</td></tr><tr><td>Zetamine</td><td>MWI Animal Health</td><td>#501072</td><td>General anesthetic</td></tr><tr><td>Xylazine</td><td>Western Medical Supply</td><td>#5530</td><td>General anesthetic</td></tr><tr><td>0.9% saline solution</td><td>Western Medical Supply</td><td>N/A</td><td>General anesthetic diluent</td></tr><tr><td>Filter Paper (12.5 cm diameter)</td><td>VWR</td><td>#28320-100</td><td>Used for removal of meninges from brain tissue</td></tr><tr><td>Centrifuge Tubes</td><td>Sarstedt</td><td>#60.540.386</td><td>Disposable tubes used for dextran centrifugation steps</td></tr><tr><td>Pierce&amp;trade; Coomassie Plus (Bradford) Assay</td><td>ThermoFisher Scientific</td><td>#23236</td><td>Measurement of protein concentration in membrane preparations</td></tr><tr><td>Wheaton Overhead Power Homogenizer</td><td>DWK Life Sciences</td><td>#903475</td><td>Required for homogenization of samples</td></tr><tr><td>10.0ml glass mortar and pestle tissue grinder</td><td>DWK Life Sciences</td><td>#358039</td><td>Required for homogenization of samples</td></tr><tr><td>Hydrochloric Acid</td><td>Sigma-Aldrich</td><td>#H1758</td><td>Required for pH adjustment of buffers</td></tr><tr><td>Bovine Serum Albumin</td><td>ThermoFisher Scientific</td><td>#23210</td><td>Protein standard for Bradford Assay</td></tr><tr><td>Standard Forceps</td><td>Fine Science Tools</td><td>#91100-12</td><td>Used for dissection of brain tissue</td></tr><tr><td>Friedman-Pearson Rongeurs</td><td>Fine Science Tools</td><td>#16020-14</td><td>Used for opening skull to isolate brain</td></tr><tr><td>50 ml conical centrifuge tubes</td><td>ThermoFisher Scientific</td><td>#352070</td><td>Used for collection of brain tissue following isolation</td></tr><tr><td>Glass Pasteur Pipets</td><td>ThermoFisher Scientific</td><td>#13-678-20C</td><td>Used for aspiration of cellular debris following dextran spins</td></tr><tr><td>Ethanol, anhydrous</td><td>Sigma-Aldrich</td><td>#459836</td><td>Used for cleaning tissue grinder; diluted to 70% with distilled water</td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman-Coulter</td><td>#41121703</td><td>Used for ultracentrifugation of samples</td></tr></tbody></table>

a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels

血脑屏障 (BBB) 是一种动态屏障组织, 对各种病理生理和药理刺激反应。这些刺激导致的这种变化可以极大地调节药物向大脑的传递, 并通过扩展, 在治疗中枢神经系统疾病方面造成相当大的挑战。许多影响药物治疗的脑屏障变化涉及蛋白质的局部化和表达在内皮细胞的水平。事实上, 这种关于健康和疾病的脑屏障生理学的知识引起了对这些膜蛋白研究的极大兴趣。从基础科学研究的角度来看, 这意味着需要一个简单但健壮和可重复的方法来分离从实验动物身上采集的脑组织中的微血管。为了从新鲜分离的微血管中制备膜样品, 必须在内皮细胞中丰富样品制剂, 但在神经血管单元的其他细胞类型 (如星形胶质细胞、小胶质、神经细胞、毛细血管)。一个额外的好处是能够从单个动物身上制备样本, 以便在实验人群中捕捉蛋白质表达的真实变异性。本文详细介绍了一种用于大鼠脑微血管分离和制备膜样品的方法。通过使用四离心步骤, 在样品缓冲中加入葡聚糖, 可获得从样品中提取的微血管。该协议可以很容易地由其他实验室为自己的具体应用而调整。从该协议生成的样本已经证明, 从蛋白质分析实验中获得健壮的实验数据, 可以极大地帮助理解对生理、病理生理学和药理刺激的脑屏障反应。

实验流程为分离的大鼠脑微血管和为制备血管膜样品显示在图 1中。使用此处介绍的过程, 成功地从大鼠脑中分离出完整的微血管 (图 2A)。这些船只是在完成离心与葡聚糖, 并立即开始离心准备膜样品 (即完成步骤 4.10) 后获得。在这个图像中, 微血管被染色使用抗体对 Oatp1a4, 一个运输者已经证明是良好的表达在血浆膜的脑微血管内皮细胞2,11,13, 18。使用西方印迹分析 (图 2B), 从雌性大大鼠的微血管中提取的膜制剂被证明富含血小板内皮细胞黏附分子 molecule-1 (PECAM-1; 也称为 CD31), 一种标记蛋白用于脑微血管。PECAM-1 是一种抑制性的联合受体, 涉及 T 细胞和 B 细胞信号, 在血管内皮细胞的血浆膜高度表达。在本协议优化过程中, 采用四葡聚糖离心步骤制备的膜样品中 PECAM 富集。如图 2B所示, 使用这些不同的离心步骤制备的样品之间的 PECAM 表达式没有差别;然而, 四葡聚糖的旋转导致血管内皮转运蛋白的表达得到改善, 这表明样品中微血管的丰富。此外, 使用四 dextrans 旋转改善去除不必要的细胞成分, 表明通过减少表达的神经元标记蛋白, 如突触素18。因此, 所有后续的大脑微血管膜的准备工作都是使用四葡聚糖离心步骤进行的。在我们的西方印迹实验中, 微管成分蛋白蛋白被用作负荷控制。根据布拉德福德蛋白测定, 膜制剂通常产生的蛋白质浓度介于5.0 毫克/毫升和10.0 毫克/毫升之间。在表 1中描述了代表性的蛋白质量化结果。这些蛋白质值是根据标准曲线确定的, 它是用牛血清白蛋白 (BSA, 2.0 毫克/毫升) 作为标准 (图 3) 生成的。在布拉德福德化验中, 蛋白质样品稀释了10倍。
根据蛋白质定量, 微血管膜样品可用于蛋白质分析的生物化学方法, 如西方印迹分析, 斑点印迹, 或共同免疫沉淀。图 4A描述了一个具有代表性的 Glut-1, 用于分析来自表 1的样本的 BBB 传输蛋白葡萄糖 transporter-1。在每个膜样品中检测到这种高度表达的脑屏障蛋白, 在适当的分子量 (即预测为 54 kDa) 上, 清除单个带。每个单独车道在这个西部污点代表微血管膜样品由一个实验动物准备。用钠钾 atp 酶 (即 Na+/K+ atp 酶) 测定每条车道上的等量蛋白质, 预测分子量 = 113 kDa) 作为加载控制。如图 4B所示, 雄性大鼠在脑屏障上的 Glut-1 表达较高, 与女性相对。在雄性和雌性动物之间观察到的 Na+/K+ atp 酶的表达没有差异。对于这种密度分析, 带量化使用 ImageJ 软件。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57698/57698fig1.jpg"> 图 1: 用于隔离大鼠脑微血管和制备膜样品的程序和协议大纲.<a href="/files/ftp_upload/57698/57698fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57698/57698fig2.jpg"> 图 2: 代表的数据, 显示完整的微血管和血管内皮细胞标记蛋白的表达.A:免疫荧光染色显示 Oatp1a4 的定位, 该转运体已被证明是良好的表达在脑屏障, 在完整的大脑微血管隔离, 如本议定书所述。用特定的兔多克隆抗体对 Oatp1a4 进行 Oatp1a4 定位, 稀释1:10。二次抗体是一种荧光共轭抗兔 IgG, 使用稀释1:300。刻度条 = 7.5 微米。B:通过对特定内皮细胞标记蛋白 PECAM-1 的富集, 通过对1:100 稀释的小鼠单克隆抗体检测, 表明了微血管样品的纯度。二次抗体是一个抗鼠 IgG 在1:50,000 稀释。BH = 大脑匀浆, MV = 粗微血管分数, MVM = 脑微血管膜。<a href="/files/ftp_upload/57698/57698fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57698/57698fig3.jpg"> 图 3: 布拉德福德蛋白质检测的典型标准曲线.牛血清白蛋白 (BSA) 被用作标准, 稀释到以下浓度: 0, 0.125, 0.25, 0.50, 0.75, 1.0, 1.5, 和2.0 毫克/毫升。光度测定为 = 595 nm。每个数据点平均为每 BSA 浓度的三吸光度读数。<a href="/files/ftp_upload/57698/57698fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57698/57698fig4.jpg"> 图 4: 具有代表性的西方印迹, 显示了从离体大鼠脑微血管制备的膜样品中 Glut-1 表达的性别差异。A:印迹分析表明, 从雄性和雌性大大鼠的微血管分离制备的膜样品中 Glut-1 表达。用兔单克隆抗体在1:500 稀释时检测到 Glut-1。二次抗体是一个单克隆抗兔 IgG 在1:40,000 稀释。Na=/K+ atp 酶, 一个等离子膜标记, 和负载控制, 检测使用一个多克隆抗兔 IgG 在 1: 4万稀释。B:密度 Glut-1 在雄性和雌性大大鼠分离膜样品中的表达。结果显示为平均扫描电镜从6实验动物每治疗组。星号表示从控件统计上显著的数据点。p < 0.05 的值被认为具有统计学意义。<a href="/files/ftp_upload/57698/57698fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>动物</td>
			<td>平均吸光度</td>
			<td>调节吸光度</td>
			<td>计算浓度</td>
			<td>实际浓度</td>
		</tr>
<tr>
<td></td>
			<td>(λ = 595 毫微米)</td>
			<td></td>
			<td>(毫克/毫升)</td>
			<td>(毫克/毫升)</td>
		</tr>
<tr>
<td>男1</td>
			<td>0.7967</td>
			<td>0.3859</td>
			<td>0.768</td>
			<td>7.68</td>
		</tr>
<tr>
<td>男2</td>
			<td>0.7849</td>
			<td>0.3741</td>
			<td>0.7417</td>
			<td>7.42</td>
		</tr>
<tr>
<td>男3</td>
			<td>0.8187</td>
			<td>0.4079</td>
			<td>0.8173</td>
			<td>8.17</td>
		</tr>
<tr>
<td>男4</td>
			<td>0.7985</td>
			<td>0.3877</td>
			<td>0.7721</td>
			<td>7.72</td>
		</tr>
<tr>
<td>男5</td>
			<td>0.7943</td>
			<td>0.3835</td>
			<td>0.7628</td>
			<td>7.63</td>
		</tr>
<tr>
<td>男6</td>
			<td>0.7251</td>
			<td>0.3143</td>
			<td>0.6084</td>
			<td>6.08</td>
		</tr>
<tr>
<td>女性1</td>
			<td>0.7114</td>
			<td>0.3006</td>
			<td>0.578</td>
			<td>5.78</td>
		</tr>
<tr>
<td>女性2</td>
			<td>0.7993</td>
			<td>0.3885</td>
			<td>0.7738</td>
			<td>7.74</td>
		</tr>
<tr>
<td>女性3</td>
			<td>0.8201</td>
			<td>0.4093</td>
			<td>0.8203</td>
			<td>8。2</td>
		</tr>
<tr>
<td>女性4</td>
			<td>0.7526</td>
			<td>0.3418</td>
			<td>0.6698</td>
			<td>6。7</td>
		</tr>
<tr>
<td>女性5</td>
			<td>0.8008</td>
			<td>0.39</td>
			<td>0.7773</td>
			<td>7.77</td>
		</tr>
<tr>
<td>女性6</td>
			<td>0.7975</td>
			<td>0.3867</td>
			<td>0.77</td>
			<td>7。7</td>
		</tr>
</tbody></table>表 1: 来自雄性和雌性大大鼠的脑微血管制剂的代表性蛋白质浓度.调整的吸光度值是通过减去每只实验动物平均吸光度值的 l = 595 nm 的背景吸收量来确定的。在这种检测中, 背景吸收率被确定为0.4108。

Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

本文介绍了一种分离大鼠脑微血管及制备膜样品的方法。该协议具有明显的优势, 生产丰富的微血管样品与可接受的蛋白质产量从个别动物。样品可用于大脑微血管内皮的健壮蛋白质分析。

一种简便、可再生的新分离大鼠脑微血管膜样品制备方法

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

a-simple-reproducible-method-to-prepare-membrane-samples-from-freshly

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.1K 次观看.  University of Arizona, Tucson. 本文介绍了一种分离大鼠脑微血管及制备膜样品的方法。该协议具有明显的优势, 生产丰富的微血管样品与可接受的蛋白质产量从个别动物。样品可用于大脑微血管内皮的健壮蛋白质分析。

视频: A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

科研

医学

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

神经科学

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

新生大鼠脑组织在神经端子突形圆锥花序分布超微结构形态分析中的制备

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

生物学

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Endothelial Cell Culture from Rat Brain Microvessels: A Procedure to Isolate and Culture Endothelial Cells from Microvessels of Rat Brain

Source: Molino, Y. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/51278/setting-up-an-vitro-model-rat-blood-brain-barrier-bbb-focus-on-bbb">Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport.</a> J. Vis. Exp.&#160;(2014)
This video presents the isolation of microvessels from a rat brain using a combination of density gradient centrifugation and enzymatic digestion mehods. The isolated microvessels can be cultured in a culture flask pre-coated with collagen and fibronectin matrix to obtain endothelial cell colonies.

大鼠脑微血管内皮细胞培养:从大鼠脑微血管中分离和培养内皮细胞的程序

Source: Molino, Y. et al. Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated ...

JoVE Encyclopedia of Experiments

癌症研究

Cancers of the Nervous System

In vitro studies

Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

Source: Collins, H. Y., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57122">Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium.</a> J. Vis. Exp. (2018)
This video demonstrates a method for isolating and culturing microglia from rat brains. It entails separating microglia from other neuronal and non-neuronal cells using plates coated with a monoclonal antibody specific to microglia.

使用无血清培养基从大鼠脑中分离和培养小胶质细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare a Petri Dish for CD11b ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating Mouse Brain Vessels: A Protocol to Isolate Intact Vessels from Murine Brain Sample

Source: Boulay, A. C. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/53208" target="_blank">Purification of Mouse Brain Vessels.</a> J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a mouse&#39;s brain that constitute the blood-brain barrier (BBB) along with neurons and glial cells. The isolated vessels can be used as an in vitro model to study their role in maintaining brain homeostasis.

分离小鼠脑血管:从小鼠脑样本中分离完整血管的方案

Source: Boulay, A. C. et al. Purification of Mouse Brain Vessels. J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a ...

In vivo studies

Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue

Source: Rubio, F. J., et. al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54358">Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue.</a> J. Vis. Exp. (2016)
This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis.

Source: Rubio, F. J., et. al. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat ...