这项工作是由神经通信技术中心（CNCT），P41资源中心，由国家生物医学成像和生物工程研究所（NIBIB，P41 EB002030）和国立卫生研究院（NIH）支持。

1。准备固定液 （请参阅表定影液和缓冲器 ）。 2。准备灌注缓冲器 （请参阅表定影液和缓冲器 ）。 3。准备仪器和麻醉<ol><li>使用的水洗澡，温暖灌注缓冲至37°C。充满缓冲液的烧杯放置在出口阀。一个缓冲区填充50毫升注射器和重视固定液油管。冲洗管道多次被驱逐和撤出缓冲区。 </li><li>清除缓冲区的行，直到所有气泡在油管被淘汰。这是不能有气泡中的任何行灌注成功的关键。 </li><li>取下注射器和连接的4％多聚甲醛固定液（室温）容器。在管端，以避免气泡，同时定位到容器管，挤压管，所以，从笔底下降缓冲区突出O触点的容器内的液体表面。 </li><li>关闭出油口（针的末端）。将缓冲阀（蓝色）作为固定液阀的（白）相同的位置。这将允许只从缓冲区行流。 </li><li>打开出气口，通过A1 A3填充缓冲区行重复步骤。所有的气泡被淘汰后关闭出气口，取出注射器和连接的缓冲容器，照顾油管引入气泡。 </li><li>测试系统的能力举行抽水橡胶压力计blub的压力。通常是由于空气压缩系统中的一些阻力。 </li><li>设置在交通便利的秩序的手术工具。填充缓冲区灌注针，以消除气泡的可能性。 </li><li>手术前，氯胺酮/甲苯​​噻嗪的混合物（高达80毫克/公斤体重氯胺酮和10毫克/公斤体重甲苯噻嗪）管理，通过腹腔注射（27号针头注射器和1毫升）。必要时将执行麻醉额外的管理，在每个操作的过程中保持麻醉的手术平面。 </li></ol> 4。灌注术<ol><li>一旦动物已经达到了手术的麻醉平面，把它洋溢着碎冰的浅盘上。 （用脚趾捏反应的方法来判断麻醉深度。动物必须是反应迟钝，然后再继续）。 </li><li>通过体壁和腹壁下方的肋骨外侧切口5-6厘米。仔细分离膈肌肝脏。 </li><li>在使用隔膜弯曲，钝的剪刀做一个小切口。可以帮助你的手指的位置和压力​​的能力，以减少隔膜。 </li><li>继续沿整个长度的肋骨暴露胸腔膈肌切口。 </li><li>将沿肋骨一侧弯曲，钝的剪刀，仔细取代肺部，使某铜通过左肋吨上涨至锁骨。对侧上作出类似的削减。 </li><li>起重胸骨而去，精心修剪的任何组织，将它连接到心脏。胸骨与止血钳尖端，并将其放置在头上止血。如果处理得当，胸腺升降机远离心脏与胸骨，提供了一个明确的看法的主要血管。 </li><li>做一个小切口，使用虹膜剪左心室后端。 </li></ol><img alt="图1" src="/files/ftp_upload/3564/3564step7.jpg" /> <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3564/3564step7large.jpg" target="_blank">点击这里查看大图</a> 。 <ol start="8"><li>截止到升主动脉心室通过15号橄榄钝或尖灌注针。小费应该是通过主动脉壁可见，不应该达到的主动脉弓，臂丛和颈动脉分歧。 </li><li>使用止血钳，夹住心脏，这针固定，防止泄漏。如果需要，修改后的止血，可用于打击针尖周围的主动脉（这些止血留在原地，直到解剖开始，但是从未来的清晰度插图省略）。 </li><li>最后，使动物的右心房，用虹膜剪刀创造尽可能大出口不损害降主动脉切口。在这一点上的动物是准备灌注。 </li></ol> 5。灌注<ol><li>打开和出气口连接针座照顾，不引入任何气泡。 </li><li>泵的压力表灯泡到80毫米汞柱的压力，快速及均匀。维护此整个缓冲区输液期间的压力。启动定时器。 </li><li>调整针头角。针的角度是至关重要的，以实现最大流量（注意流量的变化与角度调整）。 </li><li>开关的buffER阀（蓝色）（200毫升），一旦缓冲区接近完成。流体应清楚。肝脏的结算是一个良好的灌注指标。肝脏应该清楚这一点。您的记录，显示时间。 </li><li>在几秒钟内，应遵守固定震颤，这应该被视为真正的固定时间。您的记录，显示时间。 </li><li>压力可以逐步提高到最高130毫米汞柱2，保持稳定的流量。 </li><li>关闭出口阀，一旦接近完成固定液。您的记录显示结束时间。 </li><li>在这个阶段应该是僵硬的大鼠。 </li><li>使用多聚甲醛，必须收集和储存处置根据贵机构的规定。 </li></ol> 6。解剖4,11 <ol><li>删除的头，用一把剪刀。 </li><li>使从颈部到鼻子的包膜沿中线切口和暴露头骨。 </li><li>剪掉剩余的颈部肌肉，使颅底暴露，除去残留的肌肉，用剪刀或rongeurs。 </li><li>放置到一侧的枕骨大孔的虹膜剪刀对尖底，仔细沿着头骨的内表面滑动的剪刀。 </li><li>接下来，延伸到后头骨表面的远端边缘切割。使对侧相同截。使用rongeurs扫清小脑周围的头骨。 </li><li>小心地将沿颅骨内表面剪刀的尖背远端后角头骨远端前缘，起重在刀刃上，你切割，以防止损害大脑。对面的重复。 </li><li>远离大脑头骨背表面剥离rongeurs。修剪掉的头骨两侧使用rongeurs以及。 </li><li>用铲，断绝嗅球和神经连接tions沿大脑的腹面。 </li><li>轻轻挑逗的大脑远离头部，修剪任何硬脑膜仍然连接大脑的头骨，用虹膜剪刀。 </li><li>取出大脑，并将其放置在一小瓶至少10倍体积的大脑本身的固定液含流体。偶尔漩涡小瓶。 </li></ol> 7。后固定及储存设备<ol><li>保持大脑中固定液24小时在4°C，偶尔纷飞。 </li><li> 24小时后，用大脑磷酸盐缓冲生理盐水交换媒体的3倍和纷飞每次。 </li><li>大脑可以储存在磷酸盐缓冲生理盐水或HBHS叠氮化钠，并保存在4°C。 </li></ol> 8。代表结果灌注成功的一个初步的指标是四肢，如鼻子，耳朵，爪子和内脏器官，如胸腺，肝化（IHC世界）结算。总值的检查结果显示大脑的血液血管（白色至淡黄色的外观）无效。这也将是真正在组织切片染色和免疫组化注定。灌注的结果最终指标是在组织超微结构的条件。1,6,7,8 <table border="1"><tbody><tr><td> 准备固定液： </td></tr><tr><td> 准备8％多聚甲醛股票 </td></tr><tr><td><ol><li>加入500毫升DH 2 O40克多聚甲醛加热到60的解决方案 - 65°C，而搅拌（不要超过65°C间，这样做可以产生不利影响免疫组化过程中的成功）。 </li><li>要清除的解决方案，减少热量和用滴管加2-3毫升1.0 M氢氧化钠。 </li><li>过滤器和存储长达1个月在4°C。 </li></ol></td></tr><tr><td> 准备0.2 M磷酸钠缓冲液，pH值7.4 </td></tr><tr><td><ol start="4"><li>磷酸二氢钠股票，增加至1 L DH 2 O27.8克的NaH 2 PO 4计 * 2 </li><li>磷酸钠二元股票，添加28.4克的Na 2 HPO 4至1 L DH 2 O </li><li>加入810毫升到190毫升的二元股票下一代的股票。 </li></ol></td></tr><tr><td> 准备4％多聚甲醛固定液 </td></tr><tr><td><ol start="7"><li>添加等份8％多聚甲醛股票0.2M磷酸钠缓冲</li><li>注：此修补程序的最好准备新鲜的进步，在不超过72小时。 </li></ol></td></tr><tr><td> 准备灌注和存储缓冲器： </td></tr><tr><td> 准备磷酸盐缓冲生理盐水，pH值7.4 </td></tr><tr><td><ol start="9"><li> DH 2 O的 1升</li><li> 9 g氯化钠</li><li> 144毫克KH 2 PO &lt;子&gt; 4 </li><li> 795毫克的Na 2 HPO 4 </li><li>检查pH值</li></ol></td></tr><tr><td> 肝素钠缓冲Hanks液（HBHS与叠氮化钠的pH值7.4） </td></tr><tr><td><ol start="14"><li> 990毫升DH 2 O的 </li><li> 7.5 g氯化钠， </li><li> 0.3Ğ氯化钾</li><li> 0.06克KH 2 PO 4 </li><li> 0.13克的Na 2 HPO 4 </li><li> 2克葡萄糖/葡萄糖</li><li> 2.4 G 10毫米HEPES </li><li> 0.1克MgCl 2的 6个部分DH 2 O的 </li><li> 0.05克硫酸镁 7件卫生署2 </li><li> 0.165克氯化钙 2件2 O的 DH </li><li> 90毫克，南三 </li><li>检查pH值</li></ol></td></tr></tbody></table> 表1。制备定影液和缓冲器。 <img alt="图1" src="/files/ftp_upload/3564/3564fig1.jpg" /> 图1。 </stronG&gt;标记元件的灌注装置。 <img alt="图2" src="/files/ftp_upload/3564/3564fig2.jpg" /> 图2。制备灌流装置一开始与固定液行。气泡冲洗管，填补迅速驱逐出境，并撤回到注射器，并通过行的缓冲带缓冲。关闭阀针的末端。没有引入气泡，放入固定液瓶的的固定液线。 <img alt="图3" src="/files/ftp_upload/3564/3564fig3.jpg" /> 图3。灌流装置制备。 1。打开阀针的末端。 2。旋转缓冲区阀（蓝色）定位流。 3。冲洗管气泡迅速驱逐和注射器撤出缓冲区和缓冲区填写。 4。关闭阀针端。缓冲瓶放入油管。压力测试，以确保系统正确的密封LY所抽的的压力表灯泡和看压力表。该仪器是现在的灌注过程中的准备。 <img alt="图4" src="/files/ftp_upload/3564/3564fig4.jpg" /> 图4。灌注装置在缓冲区交货的位置。 <img alt="图5" src="/files/ftp_upload/3564/3564fig5.jpg" /> 图5。灌注外科一，一）通过体壁和腹壁外侧切口。 b）使在膜片的切口，暴露心脏隔膜的跨越。使锁骨平行削减肋骨两侧。 C）胸骨与止血钳尖端，并将其放置在头上止血。 <img alt="图6" src="/files/ftp_upload/3564/3564fig6.jpg" /> 图6。灌注外科二。一）截止到升主动脉心室穿过灌注针。 B）固定灌注针使用一组止血钳的心脏。第二套的修改后的止血，也可用于打击主动脉周围，以防止泄漏的针尖。用虹膜剪刀作一小切口到左心室后端。 <img alt="图7" src="/files/ftp_upload/3564/3564fig7.jpg" /> 图7。与缓冲区灌注泵的压力表灯泡创建线的压力。打开阀门（1）和附加针枢纽。这是关键，以确保无气泡介绍。泵压力计灯泡，到80毫米汞柱的压力，并保持这种整个缓冲区输液期间的压力。 <img alt="图8" src="/files/ftp_upload/3564/3564fig8.jpg" /> 图8。一旦缓冲固定液灌注几乎完成（200毫升）切换缓冲阀（1）允许流动的固定液。压力可以逐步上升到最大的130毫米汞柱。 <img alt="图9" src="/files/ftp_upload/3564/3564fig9.jpg" /> 图9。解剖一）使用了一双剪刀除去头。 b）使沿中线从颈部到鼻子的皮肤切口，暴露颅骨。 C）剪掉剩余的颈部肌肉，使颅底暴露。按住头大开幕（枕骨大孔）在颅骨表面，使访问。小心地插入枕骨大孔的一对虹膜剪刀的尖底，并作出削减。对侧重复同样的动作。使用rongeurs扫清小脑周围的头骨。 <img alt="图10" src="/files/ftp_upload/3564/3564fig10.jpg" /> 图10。夹层。一）仔细沿着头骨的内表面滑动的剪刀。提起小费，你切割，以防止损害大脑。重复相同的对面。使用剥离颅骨rongeurs远离大脑。 B）插图的头骨和大脑暴露出来。 c）用锅铲在最前的位置大脑的嗅球和神经连接切断。小心地铲尖沿易于删除切断连接大脑底面。取出大脑，并将其放置在固定液小瓶。

<ol>
	<li>Cinar, O., Semiz, O., Can, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microscopic+survey+on+the+efficiency+of+well-known+routine+chemical+fixatives+on+cryosections.">A microscopic survey on the efficiency of well-known routine chemical fixatives on cryosections.</a> Acta. Histochem. 108, 487-496 (2006).</li><li>Fritz, M., Rinaldi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+pressure+measurement+with+the+tail-cuff+method+in+Wistar+and+spontaneously+hypertensive+rats:+Influence+of+adrenergic-+and+nitric+oxide-mediated+vasomotion.">Blood pressure measurement with the tail-cuff method in Wistar and spontaneously hypertensive rats: Influence of adrenergic- and nitric oxide-mediated vasomotion.</a> J. Pharmacol. Toxicol. Methods. 58, 215-221 (2008).</li><li>Ikeda, K., Nara, Y., Yamorii, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+systolic+and+mean+blood+pressure+determination+by+anew+tail+cuff+method+in+spontaneously+hypertensive+rats.">Indirect systolic and mean blood pressure determination by anew tail cuff method in spontaneously hypertensive rats.</a> Laboratory Animals. 25, 26-29 (1991).</li><li>Jacobowitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+discrete+fresh+regions+of+rat+brain.">Removal of discrete fresh regions of rat brain.</a> Brain Res. 80, 111-115 (1974).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> Journal of Neurosci. Meth. 12, 141-149 (1984).</li><li>Jung-Hwa, T. a. o. -. C. h. e. n. g., Gallant, J., Brightman, P. E., Dosemeci, M. W., A, ., Reese, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+at+the+synapse+after+delayed+perfusion+fixation+in+different+regions+of+the+mouse+brain.">Structural changes at the synapse after delayed perfusion fixation in different regions of the mouse brain.</a> J. Comp. Neurol. 501, 731-740 (2007).</li><li>Kasukurthi, R., Brenner, M. J., Morre, A. M., Moradzadeh, A., Wilson, Z. R., Santosa, K. B., Mackinnon, S. E., Hunter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+versus+immersion+fixation+for+assessment+of+peripheral+nerve+regeneration.">Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration.</a> J. Neurosci. Meth. 184, 303-309 (2009).</li><li>Lamberts, R., Goldsmith, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation,+fine+structure,+and+immunostaining+for+neuropeptides:+perfusion+versus+immersion+of+the+neuroendocrine+hypothalamus.">Fixation, fine structure, and immunostaining for neuropeptides: perfusion versus immersion of the neuroendocrine hypothalamus.</a> J. Histochem. Cytochem. 34, 389-398 (1986).</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Aspects+of+Immunohistochemistry.">Technical Aspects of Immunohistochemistry.</a> Vet. Pathol. 42, 405-426 (2005).</li><li>Shi, Z. R., Itzkowitz, S. H., Kim, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+three+immunoperoxidase+techniques+for+antigen+detection+in+colorectal+carcinoma+tissues.">A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues.</a> J. Histochem. Cytochem. 36, 317-322 (1988).</li><li>Walker, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vertebrate dissection. , (1980).</li><li>Zwienenberg, M., Gong, Q., Lee, L. L., Berman, R. F., Lyeth, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Neurotrauma.">J. Neurotrauma.</a> Monitoring in the Rat: Comparison of Monitoring in the Ventricle, Brain Parenchyma, and Cisterna Magna. 16, 1095-1102 (1999).</li></ol>

作者没有透露。

手术成功的其他注意事项： <ol><li>一旦违反隔膜，先迅速缺氧和高碳酸血症会引发不可逆转的生理变化，可能会混淆后续分析。 </li><li>当针插入夹紧到位，剩余的压力将推动血针（闪回）的基础。事实上，较大的动物可能会大力驱逐血液。这是至关重要的血液至少达到针的基础，以避免引入气泡，将提出一个障碍，完成灌注。如果没有血针基地观察，与缓冲区中删除附加到灌注钻机出口端口的针和笔芯。一旦缓冲区已通过尖端运行，针可以重新插入到心脏。 </li><li>妥善安置主动脉内针是至关重要的，它不应该达到尽可能的主动脉弓。 </li><li>如果多个灌注正在发生，它是没有必要的ţ从一开始每次O设置。用户需要有适当量的缓冲区，并在每个瓶子的固定液（200mls动物/解决方案）。如果需要的话，瓶也可以装填。最重要的方面，要考虑的是冲洗灌注线运行，从动物的设置（输出阀）。灌注完成后，这条线将在4％多聚甲醛。从缓冲瓶油管，并用50毫升注射器装满水冲洗出来的固定液。确保在一个多聚甲醛的正确处置废物收集烧杯放在出口阀。重复了三次。笔芯用同样的方法在2.3所示的缓冲。 </li><li>这种固定方法是高度适应的能力，改变缓冲区和固定其他固定剂（如电磁），根据实验的要求。 </li><li>该系统还经常被用于提取活组织。对于这个过程只需使用提供缓冲（缓冲瓶，同时还连接到整个系统被放置在一个冰桶和固定液的瓶子是空，但密封的设置）。这保证了在最佳压力冷缓冲灌注。从这里，该系统可以适应要么直接加入染料缓冲（如果数量是不是一个问题），或使用少量之间的缓冲区的缓冲区/染料溶液的注射器输液可修复染料 - 固定液的一步。 </li><li>在血管铸型的情况下，压力监测缓冲区一步是关键，然而由于铸造溶液的粘度和巩固性质无论是50ml注射器必须直接交付或一次性的二次附件仪器（油管，阀门和控股用于容器）与当前的系统接口。我们实验室的血管铸型之前灌流装置的设计和制造。因此，我们可以没有意法半导体吃，压力测量，将在交货地点的精确测量。的数字可能要进行调整，以实现粘性流体的成功交付。 </li></ol>

<table border="1"><tbody><tr><td> 设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 麻醉剂： </td></tr><tr><td>氯胺酮/甲苯​​噻嗪的混合物（麻醉可能会有所不同每个实验室/机构） </td><td> ketaset </td><td> NDC 0856-2013-01 </td><td> 10 ml小瓶</td></tr><tr><td></td><td></td><td></td><td> 手术治疗： </td></tr><tr><td>针尖，27遗传×1.25“ </td><td>物资服务</td><td> 25251 </td><td></td></tr><tr><td>大钝/钝弯剪刀（〜14.5厘米） </td><td>罚款科学工具</td><td> 14519-14 </td><td></td></tr><tr><td>直虹膜剪刀</td><td>罚款科学工具</td><td> 14058-11 </td><td></td></tr><tr><td&gt;标准镊子</td><td>罚款科学工具</td><td> 11027-12 </td><td></td></tr><tr><td>一双罚款（格雷夫）镊子</td><td>罚款科学工具</td><td> 11050-10 </td><td></td></tr><tr><td> 1大止血钳 - 弯或直（〜19厘米） </td><td> surgicaltools.com </td><td> 17.21.51 </td><td></td></tr><tr><td> 2标准的止血钳 - 直锯齿（14厘米） </td><td>罚款科学工具</td><td> 13013-14 </td><td></td></tr><tr><td> 1修改止血钳（15号洞，通过尖端提交） </td><td>罚款科学工具</td><td> 13013-14 </td><td></td></tr><tr><td> 15号钝或尖橄榄针（血流针） </td><td> fisnar </td><td> 5601137 </td><td></td></tr><tr><td></td><td></td><td></td><td> 灌注 ： </td></tr><tr><td> HyPerfusion系统或相当于&lt;/ TD&gt; <td></td><td></td><td></td></tr><tr><td>磷酸盐缓冲盐水，pH值7.4 </td><td></td><td></td><td></td></tr><tr><td>在0.1 M磷酸缓冲液，pH值7.4的4％多聚甲醛</td><td></td><td></td><td></td></tr><tr><td>浅的玻璃或塑料托盘，约10“×10” </td><td></td><td></td><td></td></tr><tr><td>碎冰</td><td></td><td></td><td></td></tr><tr><td>水浴（37℃） </td><td></td><td></td><td></td></tr><tr><td>定时器</td><td></td><td></td><td></td></tr><tr><td> 50毫升注射器</td><td>论文产业</td><td> NPMJD50LZ </td><td></td></tr><tr><td></td><td></td><td></td><td> 剖析 ： </td></tr><tr><td>霸IR标准的锐利/钝直剪刀（12厘米） </td><td>罚款科学工具</td><td> 14054-13 </td><td></td></tr><tr><td>中等弯曲或直rongeurs的（14-16厘米）或颅骨去除钳子</td><td>罚款科学工具</td><td> 16020-14 </td><td></td></tr><tr><td>直虹膜剪刀（〜9厘米） </td><td>罚款科学工具</td><td> 14058-11 </td><td></td></tr><tr><td>微型抹刀（双2“平面的两端，一圆形，锥形1/8”） </td><td>罚款科学工具</td><td> 10091-12 </td><td></td></tr><tr><td></td><td></td><td></td><td> 后固定及贮存： </td></tr><tr><td> 50毫升玻璃瓶</td><td></td><td></td><td></td></tr><tr><td> 40毫升肝素钠缓冲Hanks液与叠氮化钠（HBHS）（90毫克/升） </td><td></td><td></td><td></td></tr></tbodY&gt; </table>

whole animal perfusion fixation for rodents

固定的目标是迅速且均匀地保持在生命的样态组织。而直接放在组织在固定液工程小块组织，像完整的大脑，造成浸泡固定的问题更大的标本的固定液，因为没有达到该组织的所有地区在同一速率5,7。通常，在缺氧的变化开始前组织可保留12。直接通过循环系统灌注固定液的优点是化学能迅速到达使用天然的血管网的有机体的每一个角落。以最有效地利用循环系统，必须采取符合生理压力3。重要的是要注意，生理压力是依赖于所使用的物种。灌注固定的技术取决于被固定的组织，该组织将如何处理录制后。在这个视频我们描述了一个低成本，快速，控制和统一的固定程序，用4％多聚甲醛灌注，通过血管系统：通过对大鼠的心脏，以取得最好的免疫组织化学脑可能保存。这项技术的主要优点（与地心吸力系统）是最有效地利用循环系统。

Watch this Scientific Journal Video about Whole Animal Perfusion Fixation for Rodents at JoVE.com

Whole Animal Perfusion Fixation for Rodents

在这里，我们描述了一个低成本，快速，控制和统一的固定程序，用4％多聚甲醛灌注，通过血管系统：通过对大鼠的心脏，大脑获得尽可能最好的保存。

整体动物灌注固定鼠害

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

whole-animal-perfusion-fixation-for-rodents

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

381.4K 次观看.  University of Michigan. 在这里，我们描述了一个低成本，快速，控制和统一的固定程序，用4％多聚甲醛灌注，通过血管系统：通过对大鼠的心脏，大脑获得尽可能最好的保存。

视频: Whole Animal Perfusion Fixation for Rodents

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管闭塞/低血压：全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

科研

医学

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

神经科学

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

一种基于微 ct 的小动物脑损伤和定位电极表征方法

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

基于增强子的表达构建体在小鼠 大脑中的 AAV部署

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

在小鼠中建立下腹部器官和后肢的原点闭路灌注

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

小鼠大脑的自由漂浮免疫染色

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. 
To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated &#945;-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated &#945;-synuclein is demonstrated in a mouse model of Parkinson's disease. This work also comprehensively describes the transcardial perfusion, dissection, tissue freezing/embedding, and frozen sectioning steps.

一种重力喂养的经心灌注方法，用于小鼠中枢神经系统的组织学分析

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

<meta charset="utf8"></head><body>使用 Neurolucida 360 进行树突状脊柱分析的方案

Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed on dendritic spines. Dendritic spines are structures of great interest in research centered around synaptic plasticity, neurodevelopment, and neurological and psychiatric disorders. Dendritic spines undergo structural modifications during their lifespan, with properties such as total spine number, dendritic spine size, and morphologically defined subtype altering in response to different processes. Delineating the molecular mechanisms regulating these structural alterations of dendritic spines relies on morphological measurement. This mandates accurate and reproducible dendritic spine analysis to provide experimental evidence. The present study outlines a detailed protocol for dendritic spine quantification and classification using Neurolucida 360 (automatic three-dimensional neuron reconstruction software). This protocol allows for the determination of key dendritic spine properties such as total spine density, spine head volume, and classification into spine subtypes thus enabling effective analysis of dendritic spine structural phenotypes.

<meta charset="utf8"></head><body>作者聚焦:优化树突状棘分析，实现海马 CA1 顶端树突的平衡手动和自动评估

Dendritic spines are post-synaptic compartments of most excitatory synapses. Alterations to dendritic spine morphology occur during neurodevelopment, aging, learning, and many neurological and psychiatric disorders, underscoring the importance of reliable dendritic spine analysis. This protocol describes quantifying dendritic spine morphology accurately and reproducibly using automatic three-dimensional neuron reconstruction software.

使用自动三维神经元重建软件进行树突状脊柱定量

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed ...

整体动物灌注固定鼠害

摘要

探索更多视频

2血管闭塞/低血压：全脑缺血模型大鼠

脑植入的微型器件及周围组织的完整的组织学特性研究

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

一种基于微 ct 的小动物脑损伤和定位电极表征方法

基于增强子的表达构建体在小鼠大脑中的 AAV部署

在小鼠中建立下腹部器官和后肢的原点闭路灌注

小鼠大脑的自由漂浮免疫染色

一种重力喂养的经心灌注方法，用于小鼠中枢神经系统的组织学分析

使用自动三维神经元重建软件进行树突状脊柱定量

整体动物灌注固定鼠害

摘要

探索更多视频

2血管闭塞/低血压：全脑缺血模型大鼠

脑植入的微型器件及周围组织的完整的组织学特性研究

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

一种基于微 ct 的小动物脑损伤和定位电极表征方法

基于增强子的表达构建体在小鼠 大脑中的 AAV部署

在小鼠中建立下腹部器官和后肢的原点闭路灌注

小鼠大脑的自由漂浮免疫染色

一种重力喂养的经心灌注方法，用于小鼠中枢神经系统的组织学分析

使用自动三维神经元重建软件进行树突状脊柱定量

基于增强子的表达构建体在小鼠大脑中的 AAV部署